SYNTHESIS OF A TRISACCHARIDE CORRESPONDING TO TEIE
GLYCOINOSITOLPHQSPHOLIPIDOLIGOSACCHAIRIDES OF THE
PROTOZOAN TRYPANOSOMA CRUZ.
Paula Naomi Brown
BSc. Combined Honours in Chemistry and Biochemistry
Dalhousie University, Halifax, Nova Scotia, 1996
THESIS SUEiMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR. THE DEGREE OF
MASTER OF SCIENCE
in the
Department of Chemistry
O Paula Naomi Brown 1998
SIMON FRASER UNIVERSITY
November 1998
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ABSTRACT
The trisaccharide ~-Galf-B-(1+3)-D-Manp-a-(l+2)-a-BManp
is found as a terminal
unit in the glycoinositolphospholipidoligosaccharides of the protozoan Tiypanosoma
cruzi, the causative agent of Chagas' disease. We describe here the synthesis of such a
unit for use in Mmunochemicaf studies. The selective activation of acetylated phenyl 1seleno-B-D-galactofiuanoside(2) over a thioglycoside acceptor with NIS/TfDH gave the
protected ~-Galf-P-(1+3)-a-D-Manp
as its ethyl thioglycoside (5). The synthesis of the
protected trisaccharide (6) was then achieved by direct activation of 5 with NISITIOH
and methyl 3,4,6-tri-0-benzyl-a-D-mannopyranoside
as the glycosyl acceptor.
Deprotection was accomplished by hydrolysis of the acyloxy substituents followed by
hydrogenolysis of the remaining protecting groups to yield the pure deprotected
as its methyl glycoside.
trisaccharide, ~-Galf-P-(1+3)-~-Manp-a-(1+2)-a-~-Manp,
BnO
This thesis is dedicated to
A unt Joyce,
who is an inspiration to me,
My Parents
for their boundless encouragement,
Peter,
for his guidance and support,
Michael,
for ahyays being able to make me m i l e ,
and Blair Johnston,
for sharing his k n d e d g e andcfi.iendrhip.
ACKNOWLEDGMENTS
1 wouid like to express my sincere gratitude to my senior supervisor Dr. B. M. Pinto, for
providing the oppominity to do this research, as well for exhibithg patience at the best of
times and restraint at the worst (sonry about all those grey hairs).
I would also like to th&
the following people:
Blair, for introducing me to carbohydrate synthesis, NMR, and the PUB crew.
Karla, for showing me the ropes and providing moral support.
Maggie, for always coming up with the hardest questions, and the "wickedest" jokes.
Oscar, Gerardo, and the Undergrads, for providing such an interesthg work environment.
Fred C l h , the Wizard, for helping me maintain a friendship with the computer in our lab.
Marcy Tracey,for recording numerous NMR spectra.
M. K. Yang, for perfonming the microanalyses.
My parents, for their encouragement and support, which included 2:00 am phone c a b .
Pete and Mike, for being the best brothers in the world.
The SFU Pub and the people therein.
TABLE OF CONTENTS
Approval Page
..............................................................................................u..
Abstract .....................................................................................................
iü
Dedication .................................................................................................
iv
Acknowledgments
...................................................................................... v
Table of Contents .................................................................................. vi
List of Tables .............................................................................................
viiï
List of Figures ..............................................................................................
ix
List of Abbreviations
...............................................................................xi
1. Introduction
1 . 1 Background
........................................................................................1
1.1.1 Biologiçal Signifïcance of Carbohydrates ................................ 1
1.1.2 Glycoconjugates in Trypanosomatids ...................................... 3
1.2 Chemical Synthesis of Oligosaccharides
............................
.
................ 8
1.2.1 Protection 1 Deprotection in Oligosaccharide Synthesis ........... 8
1.2.2 Glycosylation Methods for Pyranosides .................................. 10
1.2.2.1 Stereoselectivity ....................................................... 10
1.2.2.1a 1,2-tram-glycosylations .............................. 10
1.2.2.lb 1,2-cis-c~-D-giycosylations........................... 1 1
1.2.2.1c 1, ~ - c z s - P - D - ~ ~ G o...........................
s ~ ~ ~ ~ ~ o ~ s 13
1.2.2.2 Activation of the Anomeric Center
1.2.2.2a Glycosyl Halides
1.2.2.2b Thioglycosides
1.2.2.2~Esters
...........................
......................................... 15
............................................ 16
..........................................................
1.2.2.2d Orthoesters
15
16
................................................. 17
1.2.2.2e Glycosyl T~ichloroacetimidates................... 17
1.2.2.2f n-Pentenyl Glycosides
1.2.2.2g Glycals
................................. 18
........................................................19
1.2.2.2h Selenoglycosides ......................................... 20
1.2.2.3 Selective Activation .............................................
1.2.3 Glycosylation Methods for Furanosides
1.3 Synthetic Objectives
20
..................................23
..............................................................................24
1.3.1 Disaccharide Synthesis ............................................................ 25
1.3.2 Trisaccharide Synthesis ........................................................ 26
.
2 Results and Discussion
2.1 Synthesis and NMR Analysis of Disaccharide 5
.....................................27
2.2 Synthesis and NMR Analysis of Trisaccharide 6
..................................
32
2.3 Synthesis and NMK Analysis of Target Trisaccharide 1 ......................... 37
.
3 Experimental
3.1 General Methods ...................................................................................
42
3.2 Specific Procedures ............................................................................43
.
4 References
.............................................................................................
vii
55
LIST OF TABLES
Table 1. The nine genera of trypanosomatids............... .................................. . 5
Table 2. Common protecting groups and their methods of attachrnent and removal... 9
LIST OF FIGURES
Figure 1. Cornpanson of structures of GPIs fiom parasite proteins (Leishmania
mnjor Gp63. and T. cruzi 1G7 antigen) and a mi>mire of yeast
glycoproteins............................................................................................ 4
Figure 2. Structure of the protein-fiee GPI of T~ypanosomac w i........................... 6
Figure 3. GaFcont aining GIPLs from Leishmania. L. sarnueli. and E. schaudinni .. 7
Figure 4. 1,2~trarzr
.and 1,2.cis.glycopyranosides..
............................................. 10
Figure 5. Neighbouring group participation in a glycosylation reaction..................... 1 1
Figure 6. Mechanisms for syntheskhg 1.2~cis.glycosides. a. via the in situ
anomerization procedure and b . by use of participating solvent to
favour P-glycoside formation................................................................... 12
Figure 7. Preparation of P-D-mannopyranosidesby insoluble silver catalysts........... 13
Figure 8 . Formation of a P-D-mannopyranoside by intramolecdar inversion............. 14
Figure 9. Synthesis of P-D-mannopyranosidesvia interna1 delivery............................14
Figure 10. The use of orthoesters in oiigosaccharide synthesis................................. 17
Figure 11 . Preparation and glycosylation of glycosyl trichloroacetimidates.............. 18
Figure 12. The use of n-pentenyl glycosides in oligosaccharide synthesis................. 18
Figure 13. The use of glycals in oligosaccharide synthesis...................................... 19
Figure 14. An alternative approach to P-glycoside formation using glycals............... 19
Figure 15. Preparation and glycosylation of a phenyl selenoglycoside...................... 20
Figure 16. Glycosylation of armed and disarmed n-pentenyl glycosides................... 21
Figure 17. Relative reactivities of glycosyl donors for selective activation............... 23
Figure 18. Structure of the target trisaccharide........................................................ 25
Figure 19. The structures of the protected monosaccharides 2.3. and 4. to be
used as building blocks towards the synthesis of the protected
oligosaccharides 5 and 6...................................................................... 26
Figure 20. The synthesis of the monosaccharide donor 2......................................
28
Figure 21 . The synthesis of the monosaccharide acceptor 3.................................... 29
Figure 22. Synthesis of the protected disaccharide 5.............................................
30
Figure 23 . 400.13 MHz 2-D 'H NMR COSY spectnun of the disaccharide 5.......... 31
Figure 24. The synthesis of the monosaccharide acceptor 4..................................
33
Figure 25 . The synthesis of the protected trisacchande 6........................................ 34
Figure 26 . 400.13 MHz 2-D 'H N M R COSY spectrum of the trisaccharide 6......... 35
Figure 27. 400.13 MHz 2-D 'H NMR TOCSY spectnun of the trisaccharide 6....... 36
Figure 28. Deprotection of 6 to yield the partially deprotected trisaccharide 13..... 37
Figure 29. Deprotection of 13to yield the target trisaccharide 1............................. 38
Figure 30. 400.13 MHz 2-D 'H NMR NOESY spectnim of the trisaccharide 1..... 39
Figure 3 1 . 400.13 MHz 2-D 'H NMR TOCSY spectrum of the trisaccharide 1..... 41
LIST OF ABBIRIEVIATIONS
Ac
Acetyl
Ac20
Acetic anhydride
AcOH
Acetic acid
AgOTf
Silver trifluoromethanesulfonate
ATP
Adenoshe triphosphate
BF3-OEt2
Boron trifluoride etherate
Bn
Ben4
Bz
Benzoyl
COSY
Correlated spectroscopy
DMF
N, N-dimethylformamide
D
4-(N,N-dirnethy1amino)pyridine
W
DMTST
Dimethyl(methy1thio)sulfonium triflate
EtOAc
Ethyl acetate
EtOH
EthansI
Ga1
Galactose
GPL
Glycoinositolphospholipid
Glc
Glucose
GlcNAc
N-acetylglucosamine
GPI
Glycophosphatidylinositol
IDCP
Iodonium di-sym-collidine perchlorate
Man
Mannose
Me
Methyl
4-Me-DTBP
4-Methyl-2,6-di-t-butylpyridine
MeOH
Methanol
NBS
N-bromosuccinimide
NIS
N-iodosuccinimide
NMR
Nuclear magnetic resonance
NOBF4
Nitrosyl tetrafiuoroborate
NOESY
Nuclear Overhauser effect spectroscopy
PhSeOTf
Phenyl selenenyl tritluoromethanesulfonate
TBAHS
Tetrabutylammoniumhydrogensulfate
TESOTf
Triethylsilyl trifluoromethanesulfonate
TfuH
Trifluoromethanesulfonic acid
TMSOTf
Trimethylsilyl trifluoromethanesulfonate
TOCSY
Total correlation spectroscopy
p-TSA
para-Toluenesulfonk acid
xii
1. Introduction
1.1 Background
1.1.1 Biological Significance of Carbohydrates
The structural diversity of carbohydrates accounts for their ability to play a crucial
role in a vast array of biological processes. Carbohydrates have classically been
recognized as a medium for energy stores, fbels, and metaboiic intemediates. They also
play an important role in maintainhg structural integrity whether as components of
nucleic acids, constituents of plant and bacterial ce11 walls, or in the exoskeleton of
arthropods.
Energy is stored as starch in plants and as glycogen in animals. These are
carbohydrate polymers called polysaccharidesthat can rapidly be rnobilized when there is
a demand for energy. There are two components of starch, amylose which consists of a( 1-4)- linked glucose residues, and amylopectin which has about one a-(1-6)
linkage per
thirty a-(1-4) linkages. In plants, carbohydrates are built up fi-om COzand H20 by
photosynthesis and are involved in the conversion of CO2 into other organic compounds.
Animals are unable to syntheske carbohydrates £rom CO2 and rely on plants for their
~UPP~Y.
Glycogen is also a branched polymer of glucose residues linked by ~ ( 1 - 4 )
glycosidic bonds with branches forrned by a-(1-6) glycosidic bonds which occur about
once in every ten units. These branches serve to increase the solubility of glycogen and
make it more readily rnobilizable. Ingestion of energy sources such as starch, lactose, and
sucrose results in the absorption of their constituent monosaccharides (mainly glucose)
into the bloodstream. Glucose is then transported across cell membranes of target tissues
where it is broken down to obtain energy by glycolysis. The first step in glycolysis is the
conversion of glucose to glucose-6-phosphate, a reactant in several cellular pathways
including the production of ATP (adenosine triphosphate) and acetyl-CoA, b o t . essential
metabolic intermediates.
The structural importance of carbohydrates is demonstrated by the crucial role
they play as part of the nucleic acids. Ribose and deoxyribose form part of the structural
framework of RNA and DNA, respectively. The conformational flexibility of these sugar
rings is important during storage and expression of genetic information. Polysaccharides
are also structural elements, as in the ceîl walls of bacteria and plants. Cellulose, the
major structural polymer of plant ce11 walls, consists of glucose joined by P-(1-4)
linkages. These P linkages give rise to long straight chains that form fibds with hi&
tensile strength. The exoskeletons of insects and crustacea contain chitin, which consists
of N-acetylglucosamine residuesjoined by a P-(1-4) linkage. Chitin forms long straight
chains that serve a structural role s W a r to that of cellulose. In contrast, the a linkages in
starch and glycogen lead to open helices, in keeping with their roles as mobilizable energy
stores.
The common perception of carbohydrates has long been limited to their energy
and structural functions. Recently, it has been determined that carbohydrates on the cell
surface are key participants in biological recognition events.' Molecules containing
complex carbohydrates covalently linked to proteins (glycoproteins) or lipids (glycolipids)
are cailed glycoconjugates. These glycoconjugates often occur as components of cell
membrane^.'^
It has been demonstrated through research in many disciplinesthat the
biological information in these substances is often camed by the carbohydrate
cornPonent."
59
Glycoconjugates on the host epithelia function as receptors for vimses, bacteria,
and toxins in host-patbogen
interaction^.'^^
Research in the area of immmology has
shown that specific carbohydrate structures and complementary carbohydrate binding
proteins (known as selectins) are important mediators of lymphocyte recirculation,
activation, and inflanmatory responses."
the study of tumor cells it has been
demonstrated that alteration of glycosylation or expression of lectins has an innuence on
malignant behaviorm9The oligosaccbande structures of glycoconjugates expressed on the
d a c e of erythrocytes are known to be antigenic detemhants of the human blood group
system.Io The role of glycoconjugates Hi their biological systems are as diverse as
carbohydrates themselves; these range fiom purely structural roles to mediators in various
cellular interactions.
'* ''
6*
1.1.2 Glycoconjugates in Trypanosumatids
Many gIycoconjugates are anchored to the plasma membrane via
glycosylphosphatidylinositol (GPI) anchors. The GPI anchors of plasma membrane
proteins have been detected in organisms ranging fiom yeast to man, but occur with a
much higher fiequency in lower eukaryotes. l2?l3 AU the GPI anchors which have been
characterized to date contain an identical ethanolamine-phosphate-Man-a-(1-2)-Man-a-(1-
6)-Man-a-(l-4)-GlcN-a-(l-6jmy~hositol
backbone (Figure l), suggesting that this
sequence is likely to be conserved in all GPI anchors.
In protozoa, GPI anchors have been widely studied in their role as anchors for
ceîl surface proteins. Although some of the plasma membrane proteins of protozoa use
trammembrane polypeptide anchors, most of the major cell-surface proteins are GPIanchored. l3 This is most pronounced in many parasitic protozoa, particularly the
kinetoplastida. Kinetoplastida, of the family Trypanosomatidae, are flagellate protozoa
characterized by possession of a DNA-containing cytoplasmic organelle called a
kinetoplast. They are obligatory parasites of a vast array of k
g hosts, fiom protozoa to
man, but mainly insects.14 The developmental stages of Trypanosomatids are defined
according to body shape, emergence of the flagellum, and the relative positions of the
kinetoplast and nucleus. l5 These developmental stages are the basis for classification of
the nine genera of Trypanosomatids (Table 1). The characterization of cell surface
gIycoconjugates in the various developmental stages of these parasites is important as
there is evidence that several of these parasite-specific structures are essential for the
sumival and i d e c t ~ t yof the parasite. Some of these are known to be âirectly involved
in parasite protection
13* l6
or specific host-parasite interactions.I7,18
1 Host (vector)
1
2
3
4
5
6
.
.
T~panosoma
Leishmania
Endottypamm
Phytomonas
Herpetomonas
Crifhidia
BIastocrithzdia
Leptomonas
Rhynchoidomona;r
.
vertebrates (insects)
vertebrates (insects)
vertebrates (insects)
plants (phytophagus insects)
insects
insects
insects
insects and protozoa
insects
-
I
-
Table 1. The nine genera of trypanosomatids. l5
Several protozoa also synthesize unique GPI derivatives which are not covalently
linked to protein or rnodified by additional glycoconjugates. These low molecular weight
structures, referred to as glycoinositolphosphoiipids(GIPLs) are included as members of
the GPI family by vimie of the core sequence ~an-a(l-4)-~lc~-a(l-6)-m~o-inositol.'~
In several trypanosomatid parasites these glycolipids have been found t o be the major
cellular glycoconjugates. Trypanmoma cruzi, the causative agent of Chagas' disease
(South Amencan trypanosomiasis), has three developmental stages during its lXe cycle. lg
During the insect dwelling epimastigote stage of the parasite T. cruzi the most abundant
cell surface glycoconjugate is a GIPL.~'.21 This GIPL, formerly referred to as a
lipopeptidophosphoglycan(LPPG),"
was the first glycolipid to be isolated fiom a
trypanosomatid species that is related to the structure of the protein-bound GPI anchors.
The GIPL of T. cruzi contains the same tetrasaccharide core sequence as the
protein-bound GPI anchors, but diverges fiom the protein anchors beyond this sequence.
The GIPL contains up to two additional j3-galactofuranose residues and there is a 2aminoethylphosphonicacid group located at the C-6 position of the glucosamine. The
lipid moiety is a ceramide (Cer) containhg sphinganine and N-linked lignoceric (C24:0) acid
(Figure 2), instead of the alkylglycerol found in the protein an ch or^.^^^^^ The
oligosaccharide of this glycoconjugate is particularly interesthg due to the presence of the
galacto k a n o syl moiety .
Figue 2. Structure of the protein-fiee GPI of Tlypanosoma cnrzi
"?14
Galactofiiranose has also been isolated fiom another class of GlPLs found in
~ e i s h m a n i a . These
~ ~ GIPLs differ fiom those of T. cruzi by having a glycerolipid
instead of a ceramide and the Gay-p-(1-3)-Man rnoiety as an intemal unit in the
oligosaccharide core
'
(Figure 3a). Glycoconjugates containing galactofkanose have
also been found in the GIPLs of Leptomonas samueli
" and Endotrypanurn s c h a ~ d i n n i , ~ ~
both of which contain a ceramide üpid. In the case of L. samueli a GaI$j3-(13)-Man
unit is the terminal non-reducing sugar (Figure 3b). In E. schaudinni the Galfp-(1-3)Man disaccharide was found in the middle of the oligosaccharide chah (Figure 3c). In al1
of these GIPLs galactofiiranose is found linked P-(1-3) to a-Man,either as a terminal
non-reducing unit, as in T.cruzi, or as an intemal structure, as in Leishmania. This
specifïcity suggests that a P-(1-3)-galactofùranosyltransferase is involved in the
biosynthesis, althou& a sugar donor has not been identified. l4 Thus galactofiiranose
attachment to trypanosomatid glycoconjugates could be a good target for the
development of antiparasitic dmgs.
Although galactofùranose is widely distributed in nature, mammalian
glycoconjugates contain D-galactose in o d y the pyranose form, while the fiiranose form is
restricted to bacteriaYz6h g i , " and protozoa.217
24v25 Galactofùranosyl residues have
recently been characterized in the oligosaccharides of glycoconjugates as terminal non'17 25 or as repeating unPts in
reducing units, 14,24,27 as part of the core oligo~accharides,~~~
the backbone of polysaccharides.23 Since mammalian cells do not biosynthesize
glycoconjugates containing these structural units, galactofiuanosyl compounds are
strongly antigenic.''
The glycoconjugates on the ceil surface during the infectious stage of T.c m i are
not modified with galactofiiranose, however it has been shown that the P-D-Galfmoiety is
recognized by antibodies that inhibit T. cruzi internalization into rnammalian ~ e l l s . ' ~
Recently, it was demonstrated that T.cruzi GIPLs are able to block murine T-lymphocyte
activation2' Thus, interaction between host cellular defense mechanisrns and the GIPLs
of 7'.cruzi may play a role in establishment and maintenance of chronic infection.30 The
synthesis of oligosaccharides containhg the galactofbranosyl moiety may therefore be
useful for understanding the role Galfplays in microorganisms, for studying the
biosynthesis of hanosyl containing gIycoconjugates, and for the design of antiparasitic
drugs and the treatment of diseases caused by Trypanosoma or Leishmania species.l4
1.2 Chernical Synthesis of Oligosaccharides
The chernical synthesis of oligosaccharides requires the regioselective and
stereoselective M a g e of two carbohydrate components.
Thiç is usually
accomplished by
a reaction of the anomeric carbon (C- 1) of one sugar (glycosyl donor) with a fiee
hydroxyl group on another çugar (glycosyl acceptor). The glycosyl donor must contain a
suitable leaving group at the anomeric center such that the glycosyl acceptor may displace
the leaving group by nucleopbific displacement. This requires the selective protection of
all hydroxyl groups, except that which is involved in the formation of the glycosidic Link.
As well, the process requires a promoter to assist in leaving group displacement. It is
necessary that the carbohydrate residues be protected in such a way that selective
deprotection does not interfere with other protecting groups in subsequent steps of the
oligosaccharide synthesis.
1.2.1 Protection I Deprotection in Oligosaccharide Synthesis
Carbohydrates are polyfunctional compounds usually displayhg several Werently
reactive hydroxyl groups. To accompli& an unambiguous synthesis requires the selective
protection of ali hy droxyl groups not involved in the glycosylation. Protecting groups
can be generaliy categorized as either temporary or persistent. A temporary protecting
group may be selectively removed without affecting the remainhg protecting groups such
that the intermediate formed is available for subsequent glycosylations. The persistent
protecting groups are then removed in the final step to yield the desired deprotected
oligosaccharide. There is a diverse number of protecting groups employed in carbohydrate
chemistry, and the various strategies for their employment have been thoroughly
investigated. The most frequently used protecting groups, along with their standard
methods of attachment and removal, are presented in Table 2, adapted fiom Khan and
Protection
Deprotection
Ester
Acetyl
AQO or AcCYpyridine
Benzoyl
BzCl/pyridine
Pivaloyl
Pivaloyl chloride/pyridine
Ether
Benzyl
BnBr/DMF, NaH
p-methoxybenzyl
pCH30-BnCVDMF, NaH
Allyl
CH2=CH-CH2Cl/DMF,NaH
Trityl
(C&15)3CCl/pyridine
Silyl- Si(CH&But
Bu1(CH3)tSiCVpyridine
- Si(C&I5)zBu1
~u'(C~~)~SiCl/imidazole
Acetal
Isopropylidene
(CH&C(OCH&/acetone, H?
Benzylidene
PhCH(OCH3)t/DMF,ZnC12 or l?
pMethoxybenzylidene
pCH3û-C&CHO/ZnC12
Table 2. Common protecting groups and their method of attachent and removaL3'
The importance of the selective introduction of protecting groups in
oligosaccharide synthesis cannot be overemphasized. The protecting groups chosen must
be stable to both acidic and basic conditions, yet amenable to selective removal under
conditions sufficiently mild so as not to cleave interglycosidic M a g e s . One must also
consider that protecting groups may strongly innuence the reactMty of a sugar residue.
Finally, protecting groups may also have an effect on the fùnctionality that is to be
reacted, as in neighbouring group participation.
1.2.2 Glycosylation Methods for Pyranosides
The successfùl coupling of two sugar residues requires consideration of both
regioselect~tyand stereoselecthity, and thus is a challenging obstacle in carbohydrate
chemistry. The regioselect~tyis controlled by the selective protection of OH groups as
discussed in section 2.1. However, it should be noted that there are special circumstances
when an OH may be selectively glycosylated in the presence of other OH groups. As
most monosaccharides can have either a or p configuration, most coupling reactions
resuIt in mixtures of a-and P-anomers.
1.2.2.1 Stereoselectivity
There are two general classes of glycosylation reactions: those proceeding with
neighbouring group participation to give 1,Ztram stereochemistry and those yielding 1,2cis stereochemistry without neighbouring group participation (Figure 4).
Figure 4. 1,2-trans-and 1,2-cis-glycopyranosides.P=protecting group.
1.2.2.1a 1,2-trans-Glycosylations
The a - M a g e in the D-gîuco- or D-galacto- series corresponds to the l,2-cis type
whereas the P-linkage corresponds to the 1,2-tuam type. In contrast, the 1,2-cis in Dmannose corresponds to the p-anomer while the 1,Ztram M a g e corresponds to the a-
anomer. To achieve the desired stereoselectivity the following synthetic approaches are
available. The strategy for stereospecfic glycosyl-bond formation, illustrated in Figure 5,
shows how neighbouring g o u p participation fiom an ester (acetate, bemate, or pivalate)
at C-2 gives rise to P-Glc, P-Gd, or a-ManMages. Neighbouring group-active
substituents at the C-2 position participate in the reaction by foraiing an oxonium ion
following the promoter-assisted expulsion of the leaving group. The steric influences
exerted by this five membered ring effectively block the cis-face to incoming alcohol;
thus, the nucleophilic attack occurs preferentially fiom the tram-face. Since the reaction
likely proceeds via the initial formation of an oxocarbenium ion, it is possible to obtain
l,2-tram-glycosides regardess of the initial configuration of the glycosyl donor.
"?
x \
OP +O?
$&
+
p
O L
->
-y+ -PO*^
A O A
R
PO
OR'
PO
Figure 5. Neighbouring group participation in a glycosylation reaction.
1.2.2.1 b 1,2-cis-a-D-giycosyla tions
To synthesize cc-D-linked gluco- or galactosides, it is necessary to have a nonneighbouring group-active substituent at the C-2 position of the donor. Synthesis of a-Dglycosides via nucleophilic displacernent of the leaving group in the P-halides, shouid
induce reaction with inversion of configuration at the anomenc center. However, j3-
halides are quite reactive and anomerize to the more stable a-anomers resulting in an
anomeric mixture of giycosides. This accounts for the loss in stereoselect~tyassociated
with this method.
To bypass this problem, Lemieux et a1 32 devised the in situ anomerization
procedure (Figure 6a). The method utiüzes the more stable a-glycosyl haüde donors for
the synthesis of a-glycosides. The presence of a Lewis acid catalyst such as a tetraalkylammonium halide or metal salt causes an equilibrium between a-D- and j3-Danomers. Although the equilibrium strongly favours the a-phalide, which is stabilized by
the anomeric effect, the p-anomer is kinetically more labile and reacts preferentially with
the glycosyl açceptor with inversion at C-1 to afford an a-glycoside. The equilibrium is
then restored by fiirther anornerkation of the a-haîide to the P-haîide. The
stereochemistry can also be influenced by a participating solvent, as demonstrated in
ftom a to P, by the mechanism
Figure 6b., where acetonitrile changes the select*
a.
'.y.+
PO
p
Bi
RO Br
"
p
-
%
OR
R'OH
~
p
"
p
RO O R
Figure 6. Mechanisms for synthesizing 1,2-cis-glycosides, a. via the in situ
anomerization procedure and b. by use of participating solvent to favour
f3-glycoside formation.
r
1.2.2.1 c 1,2-cis-B-~glycosylations
Both steric and stereoelectroniçfactors disfavour the formation of P-Dmannopyranosides. The in situ anomerization procedure may not be utilized since the
kineticaliy favoured reaction of the P-D-halide would lead to the formation of the WDmannopyranoside. The 1,2-cis nature of these glycosides also make them inaccessible via
neighbouring group participation. To overcome these diEculties, a number of approaches
have been investigated.
The heterogeneous catalytic procedure, employing an insoluble catalyst such as
silver silicate, was first introduced by Paulsen and ~ o c k h oIn~this
~ procedure,
~
the
stable a-D-mannosylbromide is glycosylated, in the presence of the insoluble catalyst,
with inversion of configuration to prepare P-D-mannopyranosides (Figure 7).
Precipitation of the silver halide formed prevents fùrther assistance in anomerization. This
method does not always produce stereoselective reactions; thus, isolation of the desired
p-anomer ofien involves complicated separations.
.&/am
-
P&
OR'
Figure 7. Preparation of j3-D-mmopyranosides by insoluble silver catalysts.
Another approach to the B-D-rnannopyranosides takes advantage of the fact that
mannose is the C-2 epimer of glucose; thus, inversion at the C-2 position of P-Dglucopyranosides should result in the desired product. This inversion may proceed via an
intramolecular 34 or an intemolecular 35,36,37,38 reaction pathway. For intramolecular
inversion, a j3-glycoside is fist prepared by conventional methods. Next a t d a t e group
placed at the C-2 position, which is then displaced by the phenylurethane group positioned
at C-3. This inversion of configuration at C-2 leads to the formation of a P-mannoside
(Figure 8). P-D-Mannoside synthesis may also be achieved by oxidation of the C-2
hydroxyl group, to form an dose, followed by a stereoselective reduction.39,40,41,42
*o;;y:
PO
p
;
.
*
{
o<O
,NH
TflO
Figure 8. Formation of a P-D-mannopyranoside by htramolecular inversion.
An alternative method for the synthesis of P-D-mannosides, investigated by two
separate groups, is the intramolecular aglycon delivery method. The procedure developed
by Barresi and Hindsgaul 43 involves an aglyconic alcohol coupled to a vinyl ether
positioned at C-2 of the glycosyl donor (Figure 9). The ethyl thioglycoside is then
activated with N-iodosuccinimide (NIS) and 4-methyl-2,6-di-t-butylpyndine(4-Me-
DTBP). This is accompanied by an intramolecular rearrangement which delivers the
alcohol to the p-face of the glycosyl donor stereoselectively. Stork and CO-workers
simultaneously reported a sim.üar method involving the use of a silicon connector."
TsOH (cat.)
-4V, 20 min
1. NTS (5 eq.)
4-Me-DTBP (5 eq.)
-5°C RT, 16 hr.
--
Figure 9. Synthesis of j3- mann nos ides via interna1 delivery.
1.2.2.2 Activation of the Anomeric Center
In oligosaccharide synthesis the reactivities of the glycosyl donor and acceptor
depend on several reaction parameters, including the catalyst chosen for activation, the
protecting groups used, and also the sugar residues themselves. The activation of the
anomeric center may be achieved In many dflerent ways. The more prominent
methodologies include the Koenigs-Knorr rea~tion,'~
or the use of thioglycosides,M
n-pentenyl glycosides, 50
~rtlioesters,~~
glycosyl trichloroa~etimidates,~~
esters:
and ~ e l e n o ~ l ~ c o s i d e s . ~ ~
glycals:l
1.2.2.2a
Glycosyl Haiides
The use of glycosyl halides, either chlorides or bromides, as effective glycosyl
donors was fist introduced by Koenigs and Knorr in 1901.~' This method for glycoside
synthesis involves two steps. First, the halide leaving group must be introduced at the
anomeric center to generate the glycosyl donor. This is followed by promoter-assisted
nucleophilic substitution of this leaving group. The r e a c t ~ t yof the glycosyl halide may
be varied by the choice of halogen, the protecting groups utilized and the choice of
promoter. Activation of the glycosyl halide, by the classical Koenigs-Knorr reaction, is
achieved by silver salts in the presence of a proton acceptor. This method has been
modified by the use of mercury-containing compounds as promoters and is referred to as
the Helfench rea~tion.'~The reactivity of the glycosyl halide declines fiom iodide to
fluoride, therefore bromides and chlorides are usually employed.
The Koenigs-Knorr method presents some inherent disadvantages. Equimolar
amounts of heavy metal salt promoters are required which is expensive and ofken
dangerous. Furthemore, the glycosyl halide donors exhibit low themal stability and are
highly sensitive to hydrolysis. One advantage of the glycosyl fluonde as a glycosyl donor
is its high thermal and chernical stability as compared to the low stability of other glycosyl
halides. Therefore, glycosyl fluorides can be generally purified by an appropriate
distillation or by column chromatography on siiica gel. The use of a glycosyl fiuoride as a
glycosyl donor with a fluorophilic activator, SnClz-AgC104,was fkst introduced by
Mukaiyama et. al. in 198 1 .54 Following this discovery ,Nicolaou and his CO-workers55*
56
made extensive studies of its application in the synthesis of natural products such as
a v e r m e ~ t i n . They
~ ~ also developed the usefùl preparation of glycosyl fluorides fiom
thioglycosides.
f.2.2.2b
Thioglycosides
Thioglycosides have been extensively studied as glycosyl donors, as they offer
temporary protection of the anomenc çenter and present several possibilities for
regioselective activation.57 Their stabiiity towards various protection and deprotection
procedures make them versatile glycosyl donors. Activation of thioglycosides is achieved
by the use of electrophilic promoters. The reaction of the promoter with sulfur generates
a sulfonium ion that is subsequently displaced by a glycosyl acceptor.
Several diEerent kinds of thiophilic activators have been used including methyl
t ~ i f l a t e ,dimethyl
~~
(methylthio) &onium triflate (DMTST);'
NOBF,,
59*60 and
NIS-
T ~ D H . ~62' . The disadvantages of these methods are high toxicity and dif5culty in
handling these reagents, as well as irreproducible yields (NOBF4). Recently, the use of
hypervalent iodonium reagents with Lewis acids was examined by Fukase et. al.?) This
approach has the advantage of generating the hypervalent iodonium reagents in situ and
using less than stoichiometric amounts of Lewis acids; both high yields and
stereoselect~tywere achieved.
1.2.2.2~
Esters
The use of 1-O-acylated glycosyl donors in glycosylation reactions has the
advantage of ease of preparation. The most common acyl group used to fiinctionalize the
anomeric center is the acetyl group. First developed by Helferich et.
this method
utilized TsOH or ZnCh to activate a 1-O-acetyl sugar. Several Lewis acids have since
been shown to act as effective promoters in glycosylation, these include SnC4,
FeC13,66767 trimethylsilyl triûuoromethanesulfonate (TMSOT~
)
64,65
and boron trifluoride
etherate (BF~-OE~Z).
69
Other acyl groups, such as benzoyl and p-nitrobenzoyl groups,
may also be ernployed as anomeric leaving groups when activated by FeC13,67 TMSOTG~*
'
or B F ~ - o E ~ ~ . ~
1.2.2.2d
Orthoesters
The orthoester method of glycosylation was developed by Kochetkov and coworkers 48 and employed for the synthesis of 1,2- tram glycosidic linkages (Figure 10).
The orthoester of a simple alcohol is reacted with a glycosyl acceptor in the presence of a
promoter, 2,6-dimethylpyridinium perchlorate. The tram-esterifïed orthoester is then
rearranged by treatment with the glycosyl acceptor and a catalyst (pTSA), to yield the
1,Ztram giycoside. The orthoester glycosyIation method has since been modified with
the use of 1,2-0-(1-cyanoethylidene)derivatives, activated by TrBF4 72 or A ~ O 737
T and
~
a 1,2-O-[1-[(p-methyipiienyl)thio]ethylidene] group which is activated by TrC104 74 or
NIS-T~OH.75
POPO
0 0
R'OH
R'OH
p%+
O
pop+
P-TSA
OR
OAc
Figure 10. The use of orthoesters in oligosaccharide synthesis.
1.2.2.2e
GlycosyI Trichloroacetimidates
The initial use of an imidate as a glycosyl donor was reported by Sinay in 1976,'~
however the idea was modified by Schmidt and CO-workers49 who established
t~ichloroacetimidatesas ideal for glycosylation reactions. The trichloroacetimidate
glycosyl donor is easily prepared fiom the hemiacetal by treatment with
tddoroacetonitrile in the presence of a base (Figure 11). The glycosylation reaction is
promoted by catalytic amounts of BF~-oE~~,"
T M S O T ~ ,or~ CC13CH0
~
78 under mild
conditions. The a,P stereoselect~tymay be controlled by the choice of base used for
trichloroacetimidate formation,7gas weli as by the choice of catalytic acid used for
glycosylation.77,79, HO
-
-
-
-
Figure 11. Preparation and glycosylation of glycosyl trichloroacetimidates.
n-Pentenyl glycosides
1.2.2.2f
The n-pentenyl group was k t introduced, by Fraser-Reid and CO-workers,as an
effective activating group at the anomeric center of a glycosyl d ~ n o r . ' ~n-Peutenyl
glycosides are prepared by reaction of the hemiacetal with n-pentenyl alcohol in the
presence of an acid catalyst (Figure 12). Glycosylation reactions are commonly promoted
with iodonium dicoilidine perchlorate (IDCP),~'~
81 NIS-T~DH,~~
or NIS-TE SOT^ .83 The
study of these glycosylation reactions by Fraser-Reid and his collaborators launched the
839
discovery of a new concept of "armed and disarmed" sugars (see section 1.2.3).~~,
P
i
-
=
RO
OR'
. $ . >
RO
-'"p-+'
R"
\
Figure 12. The use of n-pentenyl glycosides in oligosaccharides synthesis.
Glycals
1.2.2.2g
Glycals, 1,2-unsaturated sugar derivathes, are versatile synthetic intermediates,
especially for the synthesis of a-2-deoxy glycosides. Initially investigated by Lemieux
and CO-workers," the reaction of a glycal with an alcohol in the presence of 12, Ag salt,
'
and a base yields a 2-deoxy-2-iodo glycoside. Several other promoters, I D C P , ~86~ *NBS,
87
and NIS
'' have since been reported.
This glycosylation involves the addition of a
halonium ion and a glycosyl acceptor across the double bond of the glycal (Figure 13).
The preferentially obtained 2-deoxy-2-halo-a-glycoside is easily converted into a 2deoxy-a-glycoside by reductive dehalogenation.
Figure 13. The use of glycals in oligosaccharide synthesis.
Using this methodology, Griffith and Danishefsky developed the suifonamido
glycosylation of glycals to effectively prepare 2-amino-2-deoxy-P -glycosides.89, 90 The
synthesis of 2-deoxy-2-@henylthio)-P-glycosides has been achieved by the addition of a
phenyl sulfonate ester t o the glycal in the presence of TMSOTf
'' or the electrophilic
activation of a glycal by phenylbis(pheny1thio) sulfonim ~alt.'~'
93 An alternative
approach to P-glycoside formation is the conversion of the glycal to a 1,2-anhydro
derivative, followed by the opening of the epoxide to afford the desired inversion of
configuration at the anomenc center (Figure 14).'~
I
,OR
0-0
\/
,OR
R'OH
Figure 14. An alternative approach to P-glycoside formation using glycals.
1
1.2.2.2h
Selenoglycosides
The fust syntheses of aryl and alkyl selenoglycosides reported in the literature
were cumbersome and involved multi-step synthesis, often requiring the initial
preparation of unstable glycosyl halides.95,96,97,98,99 An easier route to phenyl
selenoglycosides was developed by Pinto et. al. "*
of Femer and Furneaux
which is analogous to the method
'O0
for the synthesis of thioglycosides (Figure 15). Furthemore,
it was demonstrated that a phenyl selenoglycoside can be selectively activated by AgOTf
and K ZC03in the presence of an ethyl thioglycoside. Phenyl selenoglycosidesare also
susceptible to activation by lDCP 'O2 and MS. 'O3 Recently, the use of glycosyl halides to
generate aryl and aikyl selenoglycosides has been re-visited by Stick and CO-workers.'O4
The procedure for generating the selenoglycoside involves treating a glycosyl bromide
with diselenides and sodium borohydride. A related approach has also been applied t o
yield telluroglycosides.104,105
ROH
G
A
OAc
M m A c O
O
OR
C
OAc
OAc
Figure 15. Preparation and glycosylation of a phenyl selenoglycoside.
1.2.2.3
Selective Activation
The observation that electronic and structural features within a pyranose ring can
innuence the reactivity at the anomeric center has led to new protocol in glycosylation
rnethodology. This concept was first realized by Fraser-Reid who discovered that
chemoselective reactions cm be achieved betweeu two different glycosides with identical
groups at C-1, where the reactive glycosyl donor is refemed to as armed and the
unreactive one as di~armed.'~The r e a c t ~ t yof the glycosyl donor is varied by
manipulation of the protecting groups, particularly the substituent at the C-2 position.
In general, armed glycosyl donors have alkoxy or deoxy groups at C-2,whereas
disarmed glycosides have electron-withdrawing substituents such as esters or halides. The
actMty of a glycoside is also dependent upon the confonnational mobility of the pyranose
ring. The torsional effects of rings with fused cyclic acetal protecting groups are
deactivating.' O 6 Glycosylation of an armed n-pentenyl glycoside with a disarmed npentenyl glycoside results in the exclusive formation of the cross-coupled product, the
self-coupled product not being formed (Figure 16). 81434
Although the amed and disarmed strategy was initially developed for n-pentenyl
glycosides, it has been applied to other glycosyl donors. Matched pairs of armed and
disarmed giycosyl donors have been estabfished for both thioglycosides 629 'O7 and 2pyridyl thioglycosides. 'O8 Danishefsky and CO-workers investigated the stereoselective
glycosylation reaction of glycals, where merentiation of the C-3 substituent is significant
for selective glycosylation. This suggests that this methodology could be of general use
for glycosylation reactions in oligosaccharide synthesis.
Armed Donor
Ac0
Cross-coupled Product
i-
Disamed Donor
Self-coupled Product
A ~ O
Figure 16. Glycosylation of amed and disarmed n-pentenyl glycosides.
There are, however, disadvantages to the armed/disarmed approach to
oligosaccharide synthesis. The requirement for armed glycosides to be O-alkylated at the
C-2 position compromises the stereoselectidy. The resulting mixtures of a-and P-
Illiked oligosacchandes are often dEcult to separate. Furthermore, the synthesis of a
target oligosaccharide oRen requires the selective removal of protecting groups.
However, this method limits the choice of protecting groups to the persistent type, such
as the activating benzyl ether substituent.
An alternative approaçh to glycosylation, involves the selective activation of one
glycoside, glycosyl X, in the presence of another, glycosyl Y. In this strategy, the
anomeric centers of gIycosyl X and gIycosy1 Y must be functionalized with different
leaving groups, where one remains latent while the other is activated. Selective activation
of glycosides was initially described by Silwanis et. al.,log who found that the r e a c t ~ t i e s
of p-X-phenyl thioglycosides could be ~ontrolledby the choice of the para substituent.
The selective activation of a methyl thio- or phenyl thioglycoside over the comparatively
inert (p-nitropheny1)-thioglycoside was demonstrated.
The effects of activating and deactivating substituents on the reactivities of para-
substituted (pheny1thio)-a-sialosides in glycosylation reactions has also been investigated.
"O
Electron-donating substituents on tbe (phenylthio-)a-sialosides resulted in active
glycosyl donors that, by promotion with NISITIDH or DMTST, were giycosylated. An
electron-withdrawing group afforded latent thioglycosides which were inert. The use of a
nitro group to inactivate a thioglycoside has the potential of becoming active towards
electrophilic promotion by conversion of the nitro group to a NN-acetyl group.
Subsequently, Mehta and Pinto 52y
'O0
demonstrated the ability to selectively
activate a phenyl selenoglycosidein the presence of a thioglycoside. Furthermore, it was
shown that glycosyl halides could be activated selectively, with AgOTf and collidine, over
phenyl selenoglycosides and that a trichloroacetimidate glycosyl donor was selectively
activated in the presence of seleno and thioglycosides by TESOTf 52 (Figure 17). This
ability to selectively activate one glycosyl donor in the presence of another has provided
greater flexibility in glycosylation reactions.
PO
PO
SePh
Collidine
PO
*goTf
PO
SR
kCO3
PO
SR
SePh
NH
Figure 17. Relative reactivities of glycosyl donors for selective activation.
1.2.3 Glycosylation Methods for Furanosides
Although the potential of different glycosyl donors bas been explored for many
different monosaccharides in the pyranose fom, relatively little is known about the
'
reactivity of fûranosyl glycosyl donors. Work by Lederkremer et. al.
l l * '12
has
demonstrated that the condensation of a suitably protected glycosyl acceptor with an
anomeric mixture of perbenzoylated D-galactofùxanose, upon activation with SnC4,
results in the formation of a glycosidic M a g e having the P configuration. This method
which is a structural
was used to synthesize the disaccharide P-~-Galf-(1-4)-~-Glcmc
~
SnC4 was also s h o w t o activate
unit in glycoproteins of T. c r ~ z i . " Recently,
peracetylated galactofuranose in glycosylation reactions. "4 Although these methods
work, they do not have the flexibitity that c m be achieved by utilinng a glycosyl donor
which can be selectively activated.
Methods for oiigosaccharide synthesis involving fùranosyl glycosyl donors have
also been demonstrated for n-pent enyl glycosides l 15*
work by McAulBe and Hindsgaul
'
and thioglycosides.
l7
Recent
describes an indirect approach to galactofuranosyl-
containhg disaccharides invoIving acyclic glycosyl donors. Although this method works,
it too does not have the flexibfity that cm be achieved by u t W g a glycosyl donor which
can be selectively activated. For the synthesis of oligosaccharides, the most convenient
strategy is to react two glycosyl units containhg different leaving groups, where one may
be preferentially activated while the other remains latent. This method allows for the
subsequent addition of another glycosyl acceptor by selective activation of the second
glycosyl unit. Thus, it is important that selective activation strategies be developed for
furanosyl donors.
A general strategy for the synthesis of O-benzylated glycofhanosyl
trichloroacetimidatesfrom D-glucose, D-mannose, and D-galactosewas developed by
Plusquellic and CO-workers.l l9 The glycosylation of the galactofiiranosyi donor, in the
presence of Lewis acids as promoters, results in the formation l,2-czs glycosidic linkages.
However, the occurrence of galactofuranose in microorganisms is as a j3-linked moiety;13
thus, synthesis of the corresponding oligosaccharides requires the ability to f o m a 1,2tram linkage. A communication regardhg the use of phenyl 1-selenoribo~anosides
as
furanosyl donors 120 prompted our laboratory to investigate the synthesis and use of a
phenyl selenugalactofùranosyl donor. The preliminary investigation of phenyl
selenogalactofuranoçyt donors has recently been published by Johnston and ~ i n t o12'.
1.3. Synthetic Objectives
The uitimate objective of this research project was to synthesize a trisaccharide
corresponding to the glycoinositolphospholipid of the protozoan Typanosoma c m i . To
realize this goal the disaccharide j3-D-Gay-(1-3)-a-D-Man could fbst be synthesized to
afford a suitabIe glycosyl donor that could then be coupled to the C-2 hydroxyl of a
suitably protected mannopyranosyl residue. The final step would be the deprotection of
the resulting trisaccharide to yield the target molecule, 1, shown in Figure 18.
OMe
Figure 18. Structure of the target trisaccharide.
1Al Disaccharide Synthesis
In all the GlPLs of T.cruzi galactofùranose is found in a P-(1-3) linkage to an
a-mannose residue, as terminal or branched components. l4 This disacchande is also
found in the GIPLs of other parasitic protozoa (Figure 3), often within the core
oligosaccharides. 13 The synthesis of the target disaccharide 5 requires the use of
galactofùranose as a glycosyl donor. As an extension of previous work fi-om our
laboratory on the selective activation of a selenoglycoside donor in the presence of a
thioglycoside a ~ c e ~ t o rphenyl2,3,5,6-tetra-0-acetyl-B-D-selenogalac~
,'~
(2)
WUbe used as a glycosyl donor. Prelimhary work, recently published by Johnston and
~ i n t o , ' ~demonstrated
'
that the synthesis of 2 and its selective activation over the
thioglycoside 3 was a viable route to the protected disaccharide. Thus, the glycosyl
acceptor 3 wiü be synthesized as an ethyl thioglycoside, selectively protected such that
the C-3 hydroxyl is available for coupling with the glycosyl donor to yield disaccharide 5.
This strategy has the advantage of producing the disacchande such that no M e r
manipulation at the anomeric center wiil be required for its use as a giycosyl donor.
1.3.2 Trisaccharide Synthesis
The disaccharide, P-D-Gay-(1-3)-D-Man, is found a-(1-2)-linked to another a - D mannopyranosyl unit. Thus, the mannopyranosyl glycosyl acceptor 4 will be selectively
protected such that the C-2 hydroxyl group is available for coupling with 5 as the glycosyl
donor. Activation of the thioglycoside glycosyl donor with NIS-TfUH in the presence of
the methyl glycoside glycosyl acceptor will yield the protected trisaccharide 6. Stepwise
deprotection will then afford the target trisacchande 1.
Building Blocks
SePh
BnO
Protected Oligosaccharides
I
BnO
Figure 19. The structures of the protected monosaccharides 2,3, and 4, to
be used as building blocks towards the synthesis of the protected
oligosaccharidas 5 and 6,
2. Resuits and Discussion
The oligosaccharides correspondmg to the glycoinositol phospholipids (GIPLs) of
the parasitic protozoan Tiypanosonza cruzi, the causative agent of Chagas' disease,
include O-P-D
-galactofhranosyl-( 1-3>a-D-mannopyranosyl disaccharides as terminal or
branched ~ o r n ~ o n e n t s .At
~ ' the terminal non-reducing end of the oligosaccharidesthis
disaccharide unit is linked a-(1-2) to an additional a-D-mannopyranosyl unit. As an
extension of previous studies in our laboratory on the selective activation of
selenoglycoside donors in the presence of thioglycoside acceptors, 52 the synthesis of
phenyl2,3,5,6-tetra-O-acetyl-~-D-selenogalactofuranoside
(2) and its use as a
galactofuranosyl donor for glycosylation of the 3-OH position of ethyl 1-thio-a-Dmannopyranoside (3) was investigated. The disaccharide 5 was used directly as a glycosyl
donor with the selectively protected glycosyl acceptor 4 for the synthesis of the protected
trisaccharide 6. The trisaccharide 6 was then deprotected to afford the target trisaccharide
1.
2.1 Synthesis and NMR Analysis of the Disaccharide 5
The first stage in the synthesis was the formation of the galactofùranosyl donor
shown in Figure 20. When a solution of D-galactose was treated wkh methanol (MeOH)
and sulfiinc acid (H2S04) a mixture of methyl glycosides was obtained. Acetylation with
acetic anhydride (Ac20), pyridine and a catalytic amount of dimethylaminopyIidine
(DMAP) afforded a mixture of tetra-acetylated methyl glycosides. This mixture was then
acetolyzed, and the desired product, penta-O-acetyl-P-D-galactofùranose(7),selectively
crystallized.12'
Re-crystallization fiom 2-propanol yielded a white crystalline solid
obtained in an overall yield of 41%. Reaction of 7 with benzeneselenol and boron
trinuoride-etherate (BF3-OEt2) l23,52 afforded the phenyl P-D-selenofuranoside.12'
Purification by flash column chromatography gave 2 as a syrup in a 74% yield. The
selenoglycoside could not be induçed to çrystahe, but was homogeneous by TLC and
NMR, and was stable for months in the fieezer.
SePh
OAc
Figure 20. The synthesis of the monosaccharide donor 2.
The anornerio configuration of 2 was c o b e d by a 'H NMR NOESY spectnun
wlùch showed substantial H-lm-3 and H-l/H-5 NOE contacts and a lack of such contacts
between H-1 and H-4. The H-1 resonance in the 'H NMR spectnun of 2 appeared as a
closely spaced multiplet at 6 5.78 instead of the expected doublet. This was proven to be
the result of long range coupling of H-1 with H-3 and H-4, as evidenced by the presence
of weak conelations of H-1 with both H-3 and H-4, in addition to the stronger H-1/H-2
cross-peaks, in the COSY spectrum of 2. There was no evidence by 'H NMR for the
presence of more than 5% of the a-anomer in the crude reaction mixture.
A glycosyl acceptor that is selectively protected such that the C-3 hydroxyl is
available for glycosylation was then required. The synthesis of such an acceptor is shown
in Figure 2 1. D-Mannose was acetylated with AczO and pyridine with a catalytic amount
of DMAP. Reaction of the peracetylated mannose with ethanethiol and BF,OEt,
resulted in the formation of the ethyl thioglycoside in the 1,2-tuam configuration.'23
Recrystallization fiom hot anhydrous ethanol (EtOH) gave ethyl2,3,4,6-tetra-0-acetyl-1thio-a-D-mannopyranoside(8)as white crystals in an overall yield of 53%. The 'H NMR
spectnun was completely assigned by fïrst order analysis of the one-dimensional
spectrum. However, the
13cNMR
data was not in agreement with that reported by
Contour et. al..lZ4 The chemical shift of C-1 was stated to be at 6 88.9 ,while we found
that C-1 was shifted at 6 82.27. This discrepancy was also noted by Garegg et. al. lZ5
whose assignment of the chemical shift of C- 1 is in agreement with our findings.
Fuithemore, Contour et. al.
124 stated that
and 6 71.2, respectively. Analysis by
the chemical shi.sof C-3and C-5 were 6 67.8
lH/13c
heteronuclear
correlation two-dimensional
NMR spectroscopy revealed that the shiR of C-3 is downfield fiom C-5 at O 69.42, while
C-5 is found at 6 68.91.
The peracetylated ethyl thioglycoside was deacetylated, then selectively protected
at the 4-OH and 6-OH by reaction with a,a-dîmethoxytoluene andp-toluenesulfonic acid
@TSA)
126
to afford the di01 (9). Recrystallization fiom ethyl acetatekexanes gave
ethyl4,6-û-benzy lidene- 1-thio-a-D-mannopyrmoside as fine white crystals in a 32%
yield. The glycosyl acceptor 3 was obtained by selective benzoylation of 9 by phase
transfer catalysis.'21
The use of tetrabutylammonium hydrogensulfate as the phase
transfer reagent together with benzoyl chloride and 5% aqueous sodium hydroxide
converted the di01 to ethyl2-0-berizoyl-4,6-0-benzylidene1-thio-a-D-mannopyranoside
3. Purification by flash column chromatography gave 3 as a clear glass in 48% yield.
EtSH / BIjOEt2
HO
OH
Ac0
OAc
AcoAC
Ac0
set
3
Figure 21. The synthesis of the monosaccharide acceptor 3.
I
SEt
Preferential2-OH substitution was verified by the d o W e I d SMof H-2 to 8 5.56
fiom 6 4.07 in the di01 derivative 9, while the chemical shiA of B 3 did not markedly
change upon benzoylation. This was M e r confirmed by the upfield shift of C- 1, found
at 6 83.45, fiom the value of 6 84.38 in compound 9, indicating a 2-OH substitution in 3.
Although the 3-O-benzoyl derivative of 9 was not observed, ethyl2,3-di-0-benzoyl-4,6O-benzylidene-l-thio-a-D-mannopyranoside was isolated in a 25 % yield. This product
was c o b e d by f i s t order analysis of the one-dimensional 'H NMR spectrum which
showed characteristic shifts of H-2 and H-3 at 6 5.78 and 6 5.76, respectively, as a result
of the deshielding effects of the benzoyl substituents.
The reaction of the galactofhranosyl donor 2 with an excess of ethyl2-O-benzoyl4,6-O-benzylidene- 1-thio-a-D-mannopyranoside 3 at room temperature, with the
assistance of NIS-TfUH was extremely rapid, and gave the protected disaccharide 5
(Figure 22). The crude disaccharide was purified by flash column chromatography to
afford 5 as a colourless foam in a 79% yield based on 2.
ff;
NIS, TfDH
SE
HO
OAc
OAc
SEt
OAc
2
3
OAc
5
Figure 22. Synthesis of the protected disaccharide 5.
The NMR spectra of the disaccharide were assigned completely through
application of two-dimensional techniques. By following the cross-peak patterns on a
COSY spectrum it was possible to ident@ the signals which correspond to a given ring.
The unambiguous assignment of one signal within a particular ring enabled the complete
assignment of signals to inchidual rings. For compound 5, the assignment of the riug
protons of the non-reducing galactofiiranonsyl sugar was straightfonvard due to the
characteristic splittmg patterns and couplmg constants. The signal at 6 5.64 in the
spectrum was assigned to H-2 of the reducing-end sugar ring, based on the expected
deshielding of H-2 d e n geniinal to an acyloxy group. Having assigned this marker signal
to the reducing-end sugar ring, all of the ring-proton signals could now be uaequivocally
assigned (see Figure 23)
. Following assignment of the 'H NMR spectnun of 5, the 13c
NMR signals were assigned by examination of the "CI'H chernical shift correlated
Figure 23. 400.13 M H z 2-D 'H NMR COSY spectnun of the disaccharide 5.
In summary, the selenoglycoside 2 has been synthesized and shown to have
potential as a versatile galactofùranosyl glycosyl donor. The selective activation of a
selenofuranoside in the presence of a thiopyranoside using NIS/TfDH has been
demonstrated and applied to the synthesis of disacchaide 5 which may be used directly as
a glycosyl donor for the synthesis of protected trisaccharide 6 or other higher-order
oligosaccharides. Attachent of an appropriate linker-am can also lead to the synthesis
of glycoconjugates.12'
Compound 5 is the protected form of the naturaily occurring
disaccharide moiety in GIPLs of T. cruzi and other parasitic protozoa.
2.2 Synthesis and NMR Analysis of the Trisaccharide 6
The disaccharide 5 was used directly as the glycosyl donor for the synthesis of
protected trisaccharide 6. The glycosyl acceptor had to be selectively protected such that
the C-2 hydroxy was available for glycosylation; this was achieved as shown in Figure 24.
D-Mannose in acetic anhydride was treated with a solution of HBr in glacial acetic acid.
Once the sugar dissolved, indicating the completion of the acetylation, more HBr solution
was introduced to afford the glycosyl b r ~ m i d e ' ~ "in~ a 77 % yield . The success of this
one-pot method for glycosyl bromide formation was codïmed by 'H NMR spectroscopy.
The chemical shift of H- 1 was found at 6 6.29, which is consistent with the literature data.
The glycosyl bromide was then treated with MeOH and 2,6-dimethyl pyridine (2,G-
lutidine) in chloroform to give 3,4,6-tri-0-acetyl- 1,2-O-methoxyethylidene-PDmannopyranose il, which was recrystallized fiom MeOWHzO in a 28 % yield fiom 10.
The ratio of the exo to endo isomer for the acetylated orthoester was found to be 1 1: 1 by
integration of the C-OCH3 signals in the one-dimensional 'H NMR spectnun.
The triacetylated orthoester 11was deacetylated, and then benzylated with benzyl
bromide and NaH to yield the corresponding 3,4,6-tribenzyl ether 12 a s a synip. The
NMR signals of the orthoacetate C-Me and C-OCH3 protons had chemical shiRs similar to
those reported in the literature,ltg and indicated an approximately 13: 1 ratio of exo:endo
isomers. The ring openhg of 12 to give methyl 3,4,6-tri-0-benyl-a-~-mannopyranoside
4 was achieved by reflux in methanolic HCl. Compound 4 was obtained as a pure symp
by flash column chromatography in a 93 % yield.
Ho
OH
A%o
HBr 1 HOAc
+
OAc
WBrl HOAc
-O
HHO
Ac0
OH
OAc
Br
NaOMe / MeOH
P
MeOH
Ac0
BnBr 1 NaH
DMF
-YMe
MeOH 1 HCI
O
BnO
.
T
reflux
Figure 24. The synthesis of the monosaccharide acceptor 4.
The reaction of an excess of the glycosyl acceptor, methyl 3,4,6-tri-O-benzyl-WDmannopyranoside 4, with the thioglycoside donor 5 at room temperature, with the
assistance of NIS-TfOH gave the protected trisaccharide 6 (Figure 25). The crude
trisacchande was purified by flash column chromatography to afford 6 as a colourless
foam in a 68 % yield based on S.
Owing to the cornpiex overlap of signals, assignment of the 'H NMR signals of 6
was facilitated by the examination of the COSY and TOCSY spectra. The chernical
values for individual ring-proton signals within a multiplet were obtained fi-omthe COSY
cross-peak pattern. Individual vicinal coupling constants were determined fiom separated
signals in the one-dimensional 'H NMR spectra.
Figure 25. The synthesis of the protected trisaccharide 6.
For compound 6, the signals due to the H-1 ring protons were identified at 6 5.29,
6 5.24, and 6 4.80. The signal at 6 4.80 was assigned to H- 1 of the reducing-end sugar
ring, based on the expected shielding of H-1 when C-1 is attached to a methyl aglycon.
By assigning the signal at 6 5.70 as H-2' of the central mannopyranosyl ring, based on the
deshielding effects of the 2'-O-benzoyl substituent, the H-1' signal of the same ring was
identified at 8 5.24 because of the strong correlation to H-2' in the 2-dimensional COSY
spectnun (Figure 26).
The signal at 6 5.29 was a singlet which is characteristic of galactofuranosyl H-1"
signals, due to smali Ji,z
values. However, caution must be used in this assignment as the
4.2
values for a-D-mannopyranosides are also relatively small. The H-2" of the
galactofuranosyl ring was unequivocally assigned to the signal at 6 4.9 1 based on the
downfield shift which is characteristic of a proton geminal to an acyloxy group. As well,
the signal occurs as a doublet ,not the expected doublet of doublets for H-2 of a
mannopyranosyl unit, which is in agreement with the tentative a s s i m e n t of the singlet at
6 5.29 to H- 1'' of the galactofUranosy1moiety.
Figure 26. 400.13 Mtiz 2-D !H NMR COSY spectrum of the trisaccharide 6.
Having assigned these signals, the remaining signals could be assigned to their
&en rings by analysis of the TOCSY spectnim which showed through-bond 'W'H
correlations in a ring. The c o n n e c t ~ t ythrough each ring in the 2-dimensional TOCSY
spectrum of compound 6 is shown in Figure 27. The use of this two-dimensionai NMR
technique coupled with the COSY spectra mabled the complete assignment of the NMR
signals. Followbg the assignment of the 'HN M R spectnun of 6, the "C NMR spectnun
was assigned with the aid of ')c/~Hchernical shifk correlated spectroscopy.
Figure 27. 400.13 MtIz 2-D 'H NMR TOCSY spectrum of the tnsacchande 6.
2.3 Synthesis and NMR Analysis of the Target Trisaccharide 1
The fkst step in the deprotection of the trisaccharide 6 was the removal of the
acetate and benzoate protecting groups as shown in Figure 28. The protected
trisaccharide was deacylated by treatment with a methanolic ammonia solution. The
solvent and volatile materials were removed under high vacuum, then the resulting crude
symp was purified by flash column chromatography to give the partiaiiy deprotected
trisaccharide 13 as a colourless foam in 89 % yield.
Figure 28. Deprotection of 6 to yield the partialIy deprotected trisaccharide 13.
The stereochemical inte&
of the one-bond
of the trisaccharide 13 was confinned by examination
IH,for the anomeric carbons. The values
coupling constants, Jisc,
for the mannopyranosyl anomeric carbons of the central and reducing sugar rings were
168.1 Hz and 170.7 Hz, respectively. This is consistent with the presence of an a-D-
configuration about the mannopyranosyl residues.130 The complex overlap of signals in
the 'H NMR of compound 13 produced a second-order spectrum which could not be
completely assigned even with the use of 2-dimensional techniques. The one-dimensional
1
H N M R spectrum indicated that the deacylation was successfül, as the signals which
correspond to acetyl and benzoyl substituentswere no longer present. The loss of the
deshielding effect afforded to the protons geminal to acyl protecting groups accounts for
the increase in complexity of the NMR spectrum.
The anomeric proton signals fiom all three rings of the trisaccharide were cas*
identified. The signal at 6 4.80 in the spectrum was assigned as K 1 of the reducing-end
mannopyranosyl rin& based on the characteristic shieldhg of H-1 by the methyl aglycon.
M e r analysis of the splitting patterns and coupling constants showed the signal at 6 4.03
was H-2", the signal at 6 5.13, which had a strong correlation to H-2" in the COSY
spectnun of 13, was assigned as H-1 " of the non-reducing fùranosyl ring. H-1 'of the
central non-reducing mannopyranosyl ring was then identified as the signal at 6 5.09,
which was conelated in the COSY spectnim to the signal at 6 4.21, assigned as H-2'.
The benzyl protecting group signals were still visible in the 'H NMR spectnun as
three sets of AB patterns, and the characteristic signal at 6 5.60 indicated that the 4,6-0benzylidene group was stiIl intact. Based on these observations, as weil as the presence of
three anorneric H-1 signals and the loss of signals correspondhg to the acyloxy protecting
groups, we were satisfied that the deprotection was successful and the resulting compound
was in fact the trisaccharide 13.
With the successfùl completion of the fkst step in the deprotection of 6, the final
deprotection reaction for the synthesis of the target trisaccharide was undertaken, as
shown in Figure 29. Compound 13was dissolved in 80 % acetic acid and hydrogenolyzed
over palladium-carbon at a hydrogen pressure of 52 psi. The desired deprotected
trisacchande 1was obtained in 96 % yield.
80% acetic acid
52 psi
I
OMe
- --
Figure 29. Deprotection of 13to yield the target trisaccharide 1.
TO confirm the stereochemistry of the deprotected trisacchsride 1,the one bond
"c-'H
couphg constants for the anomeric carbons were examhed. The
value for
the reducmg-end mannopyranosyl ring was 172.1 Hz and for the central non-reducing
was 171.4 Hz,both consistent with an a-Dconfiguration
mannopyranosyl ring JIIC,IH
about the mannopyranosyl rings. The P -D-configuration of the non-reducing
galactofiuanosyl ring was codimed by the 'H NMR NOESY s p e ç t m (Figure 30)
which showed an NOE contact between H-1 " and H-3' and no contact between H- 1"
and H-4' '. H-1 "IH-3' and H- 1"/H-2" NOE contacts were also present, confïrming the
identity of the Galf-J3-(1-3>a-Ma.n glycosidic linkage and the anomeric configuration of
the Galfmoiety, respectively.
Figure 30. 400.13 MHz 2-D 'H NMR NOESY s p e c t m of the trisaccharide 1.
The one-dimensional 'H NMR spectrum was complicated by a complex overlap of
signals. Proton signals corresponding to the three anomeric H-1s and the three H-2s were
readily identified by their chemical shifis and splitting patterns. The signal at 6 4.96 was
assigned as H-1 of the reducing mannopyranosyl ring, based on the shielding effects of the
methyl glycoside. The other two anomeric protons had signals with very close chemical
shifts, at 6 5.13 and 6 5.03, both appearing as broadened singlets. The corresponding H-2
protons were assigned by analysis of the COSY spectrum, but again both H-2's exhibited
values of 1.5 and 1.6 Hz, and J
similar chemical shifts and couphg patterns, with JBIw
HzJ.i3valuesof 3.1 and 2.9 Hz. Examination of the COSY s p e c t m enabled the
assignment of signals to the three H-3 protons, as well as the Werentiation between the
three individual spin systems. The signal at 6 5.13 could then be assigned as H-1" of the
furanosyl ring based on the H-3" assignment to the signal at 6 4.04, which had a JH3HI
value of 6.5 H i that is charactenstic of the H-3 of a galactofiiranoside. The signal at 6
5.03 was then assigned to H- 1 ' of the non-reducing mannopyranosyl ring. With the aid
of the TOCSY spectra (Figure 3 1) the individual spin systems were differentiated;
however, the complete characterization of aU the sisals was not possible due to extensive
overlap of chemical shiRs resulting in a second order s p e c t m .
AIthough complete assignment of all the signals in the 'H NMR spectnun of
compound 1was not possible, the synthesis of the target trisacchande was considered
successful. The identification of the three anomeric proton signals, and the lack of any
signals corresponding to the remaining protecting groups fiom compound 13 supported
this claim. The signals in the 13cNMR spectnim correlated with the structure of
trisaccharide 1; the microanalysis for compound 1 was also consistent with the proposed
structure in Figure 18 . Based on the evidence presented above, it can be concluded that
the synthesis of the target trisaccharide 1was achieved successfully.
Figure 31. 400.13 MHz 2-D 'HNMR TOCSY spectnim of the trisaccharide 1.
The target trisaccharide is the methyl glycoside of a naturally occurrnig
trisaccharide moiety found in the GlPLs of T~panosomacruzi, the causative agent of
Chagas' disease. Given the antigenic nature of galactofllranosyl-containing compounds,
this research has layed the groundwork for the immunochemical studies. Future work
wouid consist of synthesizing the monosaccharide acceptor 4 as the allyl glycoside to
couple with the disaccharide 5 to make the allyl derivative of trisaccharide 1. Conjugation
of the allyl glycoside of 1to solid supports or soluble protein camiers will permit the
preparation of a f i t y columns and antigens for use as immunoadsorbents or vaccines,
respectively.
3.1 General Methods
Reagents were used without M e r pudication, and solvents were distilied and
dned before use, as necessary, by literature procedures. Glycosylations were carried out
under N2. Solutions were concentrated in vacuo with bath temperatures not exceeding
50°C. Optical rotations were measured at 21°C with a Rudolph Research Autopol II
polarimeter. Melting points were determined with a Fisher-Johns melting point apparatus
and are uncorrected. Ail new compounds were characterized by microanalysis.
TLC was performed usiag aluîlllflum plates, pre-coated with Merck silica gel 60-
F254as the adsorbent. Visualization was by exposure of the dried plates to UV light or by
spraying with a solution of 1% ceric sulfate and 1.5% molybdic acid in 10% aqueous
H2SO4, and heating. Purification was achieved by flash coIumn chromatography on siîica
gel 60 (Merck, 230-400 me&) accordmg to a published procedure."'
1
H and 13cCspectra were obtained using a Bmker AMX-400 NMR
spectrometer operating at 400.13 and 100.6 MHz for proton and carbon, respectively.
Spectra were measured in CDC13 for solutions of protected compounds, unless othenvise
stated. Chemical shifts in CDCb are reported relative to extemal TMS. Where
necessary, assignments for 'H N M R were confirmed with the aid of two-dimensional
'W'H COSY spectra (COSYDFTP) in combination with TOCSY experiments. AU
13c
spectra were assigned by two-dimensional 'H/'~cheteronuclear correlation experiments
(INVBTP) using standard Bruker pulse programs and an inverse-detection, 'WX double
resonance probe. Chemical shifis and couplhg constants were obtained fiom a first-order
analysis of the spectra.
3.2 Specific Procedures.
A solution of galactose (20. log, 111.6 mmol) in MeOH (400 ml) was cooled to O OC in
an ice bath. Concentrated H2S04 (3.00 ml) was added dropwise and the reaction mixture
was allowed to warm to room temperature. The mixture was stirred at room temperature
for 2.5 h. The reaction mixture was neutralized by stining with Dowex (-OH) anion
exchange resin, fïltered, and concentrated to give a mixrture of methyl galactosides as
symp. The crude mixture of methyl galactosides was then acetylated by dissolution in
acetic anhydride (90 ml) and pyridine (1 10 ml), and addition of a catalytic amomt of
DMAP. m e r stirring at room temperature for 24 h. the Ac20 and pyridine were
removed on rotary evaporator under hi& vacuum, then CO-evaporatedwith toluene (2 x
30 ml) to yield a pale yeilow syrup. The mixture of tetra-O-acetylated methyl
galactosides was dissolved in AczO (30 ml)/AcOH (140 ml). The reaction mixture was
cooled to O OC in an ice bath while concentrated H2S04 (6.00 ml) was added dropwise.
The reaction was stirred at room temperature for 24 h. then poured over crushed ice
(-800 ml) and extracted with CH& (3 x 100 ml). The combined extracts were washed
with saturated NaHCOs (2 x 50 ml), dried over anhydrous MgS04, filtered, and
concentrated to a syrup. Selective crystallization of the desired isomer, 1,2,3,5,6-penta-
O-acetyl-P-D-galactofiuanoside(7), £kom 2-propanol yielded a white c r y s t a b e solid
(17.66 g, 41 %) : mp 95-97 OC; [alD-37O (c 2.0, CH2C12), [lit.122mp 96-97 O C ,
[all,-
41.5O (C 2.0, CH2C12)];
1
HNMR: 6 6 . 1 8 ( b r s , 1H,H-l),65.35(ddd, 1- J4,s=Js,s,4.0,J5,6b7.0Hz,
H-5), 6 5.18 (dd, 1H, J i , 2 0.7, Jz,3 2.0 HZ, H-2), 6 5-08( ddd, lH, J i , 3 0.5, J 3 , 4 5.4 Hz, H3
8 4.36 (dd, lH, H-4), 6 4.33 (dd, l& J6q6b 12.0 HZ,H-6a), 6 4.21 (dd, ll&H-6b),
6 2.129, 2.125,2. 12,2.11, 2.05 (5s, each 3H, 5 x -C(O)CH3);
13c
NMR:
6 170.44, 169.93, 169.69, 169.33, 168.96 (5 x -C(0)CH3), 6 99.07
(C-1), 6 82.10 (C-4), 6 80.55 (C-2), 6 76.27 (C-3), 6 69.21 (C-5), S 62.49 (C-6), 6 20.90,
20.72, 20.58 (3C),(5 x - c ( o ) a 3 ) .
Phenvl 2.3-5 -6-tetra-O-acetyl- 1-seleno-@-D-galactofùranoside (2)
Diphenyldiselenide(4.03 g, 12.9 m o l ) was refluxed in 50% &PO4 (40 ml) under a
nitrogen atmosphere for 3 h., then s h e d at room temperature for 13 h. After an
additional 6.5 h. at reflux conditions the reaction was cooled to room temperature, diluted
with CH2Cl2 (40 ml) and washed with cold H20(20 ml). The aqueous layer was washed
with CHzClz (2 x 20 ml). The organic fractions were collected, washed with cold H 2 0
(10 ml) and a mixture of icelsaturated NaCl (20 ml each), dried over anhydrous MgS04,
filtered, and washed with CH2Cl2 (2 x 20 ml). The pale yellow solution of benzeneselenol
and 1,2,3,5,6-penta-0-ace~l-B-pgalactofiuanoside
(10.1244 g, 25.9 mmol) was stirred
with BF3:etherate (2.6 ml, 20 mmol) at room temperature under N2 atmosphere for 18 h.
The mixture was diluted with CH2Cl2 (50 ml), poured into a mixture of icelsaturated
NaHC03 (50 ml each) and stirred until the bubbling ceased. The aqueous phase was
extracted with CHzCl2 (2 x 30 ml) and the combined extracts were washed with saturated
aqueous NaHC03 solution (2 x 30 ml) and cold water (20 ml), dried over anhydrous
MgS04, filtered, and concentrated to give the crude selenoglycoside as a light brownish
synip. Chrornatography on silica geI (1:1, hexanes-EtOAc) gave pure phenyl2,3,5,6-
tetra-O-acetyl-1-seleno-m-galactofuranoside (2) as a pale-yellow syrup (9.08 g, 72 %):
[alID
-135.5
O
(c 1.8, CH&),
[alo-152
O
(c 1.0, CHC13)];
1
H NMR: 6 7.63-7.58 (m, 2H, aromatic), 6 7.34-7.26 (m,3H, aromatic), 6 5.78
(dd, IH, J i , 2 1-89J1,3= JI,Q= 0.8 & H-l), 6 5.43 (ddd, lH, J4,5 4.0, J s ,4.5,
~ ~J5,6b7.1
HZ, H-5), 6 5.30(dd l H J ~ ~ 3 2k. ,0H - 2 ) , 65.06 (ddd, lH, J3,45.6Hz,H-3), 64.48
(ddd, lH, H-4), 6 4.31 (dd, lHJ6a,61, 11.8 Hz,H-6a), 8 4.16 (dd, lH, H-6b), 6 2.12, 2.11,
2.09, 2.04 (4s, each 3H, 4 x -C(O)CH3);
13cNMR:
6 170.38, 169.91, 169.73, 169.48 (4 x -C(0)CH3), 6 134.47 (2C),
129.09 (SC), 128.73, 128.09 (aromatic), 6 86.26 (C- 1), 6 82.03 (C-Z), 6 80.60 (C-4),
6 76.57 (C-3), 6 68.98 (C-5), 6 62.47 (C-6), 6 20.71 (2C), 20.61(2C), (4 x -C(0)CH3).
D-Mannose (34.53g, 191.6 mmol) was dissolved in Ac20 (150 ml) and pyridine (175 ml)
and stirred with DMAP (-30 mg) at room temperature for 1.5 h. The Ac20 and pyridine
were removed in vacuo, and CO-evaporatedwith toluene to yield a pale yeilow synip.
The product was dissolved in CH2C12 (250 ml) and washed with saturated NaHCOs (2 x
100 ml), 5% HCI (100 ml), saturated NaHC03 (100 mi) and H20 (100 ml), dried over
anhydrous MgSQ, filtered, and concentrated to yield a pale yellow symp. The
peracetylated mannose (15.4 1 g 39.5 mmol) was dissolved in CHC13 and cooled to O OC.
Ethanethi01 (3.00 ml, 40.5 m o l ) and BF3:etherate(9.70 ml, 79 mmol, 2 equiv.) were
added, and the reaction mixture was stirred at O OC under N2 atmosphere for 2 h. After
stirring at room temperature for 18 h. an additional 0.3 equiv. of ethanethi01 (1.00 ml)
was added to the mixture. M e r 30 min. the reaction was diîuted with CHC13(150 mi)
and washed with ice H 2 0 (100 ml), saturated NaHC03 (100 ml), a mixture of
icehaturated NaHC03 (50 ml each), cold H20 (100 ml), dried over anhydrous MgSO,,
filtered, and concentrated to yield a crude white solid. Recrystallization fiom hot
anhydrous EtOH yielded the title compound (8) as white crystals (8.26 g, 53%): mp
104-106 OC;[alD+98.5 "
(C
0.76, CHC13), [lit. 124 Dlp 101-102 OC; [alD98.7 " (C 0.9,
CWC13)I;
'HNMR: 6 5.34(dd lH.,J,,z1.6, &,33.2& H-2), 6 5.31 (dd, lH,J3,4=J45=
9.7 HZ,H-4), S 5.29(d, 1H,H-1), 6 5.26(dd, lH, H-3), 64.40(ddd, lH, Js,6,5.4,&,3,
2.3 HZ, H-5), 6 4.32 (dd, lH, J6a,6b 12.2 HZ, H-6a), 6 4.09 (dd, IH, H-6b),6 2.67 (dq,
lH, J
H 13.1
~ HZ,
~ -SCHaHbCH3), 6 2.60 (dq, lH, -SCHaHbCH3), S 2.17,2.09,2.05,
1.99 (4s, each 3% 4 x -C(O)C&), 6 1.30 (t, 3H, J 7.4 PZz, -SCH2CH3);
13CMMR: 6 170.54, 169.94, 169.69 (SC) (4 x C(0)CH3), 6 82.27 (C-1), 6 7 1.92
(C-2), 6 69.46 (C-3), 6 68.91 (C-5), 6 66.38 (C-4), 6 62.42 (C-6), 6 25.44 (-SC&CH3),
6 20.88,20.66 (ZC), 20.58 (4 x -C(0)m3), 6 14.72 (-SCH2CH3).
The peracetylated ethyl thioglycoside (3.92 g, 9.98 m o l ) was stirred in MeOH (75 ml)
with 1M NaOMe/MeOH (2.80 ml) at room temperature. After 16 h. the reaction mixture
wis neutralized by stimng with Rexyn 101 (H*)ion exchange resh. The resin was
filtered, washed with MeOH (2 x 20 ml), and the solvent removed in vacuo to give a
quantitative yield of a pale yellow synip. The deacetylated ethyl thioglycoside was
dissolved in D M . (15.O ml). a,a-Dimethoxytoluene (1.8 ml, 12.0 mmol, 1.2 equiv.) and
p-toluenesulfoniç acid (30 mg) were added and the reaction stirred under an
atmosphere at room temperature for 18 h. The reaction mixture was then neutralized by
stimng with K2CO3for 30 min.,the D M . was evaporated in vacuo, and the reaction
mixture stored at 4OC for 48 h. The crude mixture was ailowed to reach room
temperature and was then dissolved in EtOAc (300ml) and Hz0 (200 ml). The organic
fraction was washed with H20 (2 x 100 ml) and the aqueous extracts were collected and
washed with an additional 100 ml of EtOAc. The organic fiactions were combined, dned
over anhydrous MgS04, filtered, and concentrated to yield cmde white crystals (2.14 g
69%). The crystals were recrystallized fiom hot EtOAchexanes to yield the title
compound (9) as fine white crystals (1.01 g, 32 %): mp 175-176 OC; [alo1-161' (c 1.22,
CHCl3); [lit. lz5 mp 174-175 O C , [& +167.5 (c 1.22, CHC13)J;
O
1
HNMR: 6 7.56-7.33 (m, SH, aromatic), 6 5.57 (s, lH, PhCH), S 5.37 (d, lH,
6b 11.6 HZ, Hm5), 6 4.24 (dd, ZH, J&,(jb 11.6
H-l), 6 4.25 (ddd, l q J 4 , 5 9.7, Js,6r 5.0, Js,
Hz, H-6a),64.14(dd, 1H,J,,2 1.1H.H-2), 64.07(ddYlHJ2,33.4Hz,H-3),
(dd, lH,
J3,4
9.7 HZ,B4), 6 3.85 (dd, le H-6b), 6 2.68 (dq, IH, J
63.97
H13.0~HZ,~
-SCHnHbCH3), 6 2.59 (dq, 1- -SCHaHbCH3), 6 1.3 1 (t, 3H, J 7.4 HZ, -SCH2CH3);
13
C NMR: 6 137.17 (aromatic C- 1 of PhCH), 6 129.27, 128.33 (2C), 126.25
(2C) (aromatic), 6 102.28(PhCH), 6 84.38 (C- l), 6 79.17 (C-4), 6 72.36 (C-2), 6 69.13
(C-3), 6 68.62 (C-6), 6 63.45 (C-5), 6 25.07 (-SCH2CH3), 6 14.80 (-SCH2m).
The di01 (9) (202 mg, 0.65 mmol) was dissolved in C&Ch (18.0 ml) and the solution was
cooled in an ice bath. Tetrabutylammonium hydrogendate (43 mg, 0.128 mmol),
benzoyl chloride (98.1 @, 0.85rnmol, 1.3 equiv.), and 5% aqueous NaOH ( 1.40 ml) were
added and the reaction mixture was sthed for 35 minutes at 0-5 OC. The reaction
mixture was diluted with CH2C12 (20 ml), washed with H20(20ml), dried over anhydrous
MgS04, filtered, and concentrated to a clear syrup. Purification by flash column
chromatography (toluene:EtOAc, 6: 1) gave the 2-O-bemyl derivative (3) as a clear glass
~ O (c 0.84, CHC13);
(129.4 mg, 48 %): [ a ]+51.2
1
H NMR: 6 8.14-8.08 (m, 2-
[alo+49.5 (c 0.84, CHC13)];
O
O-Hin benzoyl), 6 7.65-7.36 (m, 8H, aromatic),
6 5 . 6 6 ( ~ 1, 5 PhCH), 65.56(dd, lH,J1,21.0, J2,33.4H~,H-2),65.42(d7 1H,H-1),
6 4.34-4.27 (m,3H, H-3, H-5, H-6a), 6 4.08 (m, 1- H-4), 6 3.90 (m, lH, H-6b), S 2.68
(dq, 1H,
JbHb
13.0 )4 -SCHaHbCH3),
6 2.59 (dq, lH, -SCHaHbCH3), 6 1.32 (t, 3H,
J 7.4 HZ, -SCHLCH3);
NMR: 6 166.06 (-C(O)Ph), 6 137.09 (aromatic C- 1 of PhCH), 6 133.44
13c
(aromatic C- 1 of -C(O)Ph), 6 129.91 (2C), 129.30, 128.83 (2C), 128.34 (2C), 128.22,
126.35 (2C) (aromatic), 6 102.38 (PhCH), S 83.45 (C- l), 6 79.76 (C-4), 6 74.44 (C-2),
6 68.65 (C-6), 6 67.99 (C-3), 6 64-13 (C-5), 6 25.79 (-SCH2CH3), 614.95 (-SCH2CH3).
Tlie selenoglycoside (2) (102 mg, 0.21 m o l ) and the selectively protected ethyl thio
mannopyranoside (3) (93 mg, 0.22 mmol) were dissolved in CH2C12 (4.0 ml). The
solution was stirred with fieshly activated powdered 4 À molecular sieves ( 4 . 5 g) under
an N2 atmosphere at room temperature for 15 minutes. Niodosuccinimide (5 1 mg, 0.23
mmol, 1.1 equiv.) was added followed, afier 5 minutes, by triflic acid (1 pl) via syringe.
An immediate reaction that produced a dark purple-brown colour ensued. M e r 45
minutes, triethylamine was added to quench the reaction and the mixture was Htered and
washed with CH2Ch (2 x 25 ml). The fütrate was washed with 10% aqueous NaS203(20
ml) and H20 (20 ml), dried over anhydrous MgS04, Htered and concentrated to a dark
yeUow symp. Pudication by flash column chromatography (toluene-EtOAo, 3: 1) gave
the disaccharide (5) as a colourless foam (124.0 mg, 79 %): [alD-19 O (c 1.84, CHC13);
[lit. 12' [ a ]-5~O (c 0.6, CHC13)];
1
H NMR: 6 8.11-8.09 (m, 2H,aromatic), 7.63-7.34 (m, 8H, aromatic), 6 5.64
(dd, lH., J1.2 1.3, J2,33.8HZ,H-2), 65.63 (s, lH,PhCH), 65.41 (d, lH,H-l), 65.31
(ddd, lH, J47,593.1, Js*,sa*7.2, Js*,w4.7H~,H-5'),6 5.26(~,l y H - l ' ) , 64.96(d, lH,
Jz; 3 7 1.9 HZ, H-Z'), 6 4.85 (dd, lH,
3), 6 4.33 (ddd,
54.5
=J5,6bZ
Jy,1*
6.0 HZ,H-3'), 6 4.34 (dd, lJ3, J3,4
10.0 HZ, H-
10.0 &
J5,&
4.6 Hz, H-5), 6 4.29 (dd, %
I,
Ar,&
10.0 Hz, H-6a), 6 4.28 (dd, 1- H-47, 6 4.12 (dd, lH, J6.v,6t,9 12.1 H z , H-6a9), 6 4.09
13.0
(dd, lH, H-4), S 4.07 (dd, IH, H-6b'), 6 3.92 (dd, IH, H-6b), 6 2.71 (dq, lH, JHam
HZ,-SCHaHbCH3), 6 2.64 (dq, lH, -SCHaHbCH3), 6 1.32 (t, 3H, J 7.4 Hz, -SCH2CH3),
6 2.10, 2.07, 1.94, 1.81 (4s, each 3H, 4 x -C(O)C&);
13
C NMR : 6 170.23, 169.83, 169.73, 169.04 (4 x -C(0)CH3), 6 165.53
(-C(O)Ph), 6 137.52 (aromatic C- 1 of PhCH), 6 133.32 (aromatic C- 1 of -C(O)Ph),
129.93 (2C), 129.84, 129.05, 128.48 (2C), 128.21 (2C), 126.07 (2C) (aromatic),
3.4.6-Tri-O-acety11.2-O-~~~~~~~~~~~~~~~~~~~~orose (11)
D-Mannose (16.39 g, 91 mmol) in a mixture of acetic anhydride (75 ml) and a solution of
HBr in glacial acetic acid (45 % wlv, 15.0 ml) was stirred at room temperature until al1
the solids went into solution (15 min). An additional 75 ml of HBr solution was then
introduced, and after stirring for 2.5 h. at room temperature, the clear solution was stored
at -20 OC for 18 h. The reaction mixture was wamed to room temperature then poured
over ice/water miuhire (800 ml) and extracted with CHzClz (2 x 200 mi).
The organic
fractions were collected and washed with saturated NaHC03 (2 x 60 mi) and H 2 0 (60
ml), dried over anhydrous MgS04, filtered and concentrated under reduced pressure and
repeatedly CO-evaporatedwith toluene to yield a syrup (28.79 g, 77 %). The syrupy
tetra-O-acetyl-a-D-mannosyl
bromide was dissolved in chlorofom (90 ml) and cooled in
an ice bath. To this solution 24 ml of 2,6-dimethylpyridine (56-lutidine), in absolute
methanol(140 ml), was added. After stirring for 18 h. at room temperature the solution
was concentrated to a syrup, dissolved in CHCl3 ( 90 ml), and washed with ice cold 3 %
aqueous NaHC03 (2 x 45 ml), dried over anhydrous MgS04, and fltered. The solvent
was evaporated in vacuo until a semi-crystalline residue was fomed, then CO-evaporated
with tol.uene (- 45 ml). The solid product was crystallized fiom MeOH/H20to yield the
title compound (11)as white crystals (6.40 g, 28 %): mp 106-108 OC; [alD-9.0" (c 1.00,
CHCL); ~ i tlz9. mp 109-110 OC];
exo-isomer :
'HNMR.
S 5.49 (d, 1H, J1,,2.6Hz,H-I), 6 5.30 (dd, lH, J3,4=J45=9.8Hz,
H-4)7 6 5.15 (dd, 1W,J 2 , 3 4.0 H Z 3 H-3), 6 4-61 (d, 1H.yH-2), 6 4.24 (dd, 1 5
6b
4.9, J&,
12.0 % H-6a), 6 4.14 (dd, lH, Js, 6b 2.6 Hz,H-6b), 6 3.68 (ddd, lH, H-5),
6 3.28 (s,
3H, -C(CH3)0CH3), 62.12, 2.07,2.05 ( 3s, each 3H, 3 x -C(0)CH3), 6 1.74 (s, 3H,
-C(CH3)0CH3).
NMR: 6 170.70, 170.37, 169.46 (3 x -C(O)CH3), 6 124.43 (-C(CH3)0CH3),
13c
S 97.3 1 (C-1), 6 76.54 (C-2), 6 71.22 (C-5), 6 70.55 (C-3), 6 65.48 (C-4), 6 62.27 (C-6),
6 49.84 (-C(C&)OCH3), 6 24.27 (-c(a3)ocfi), 6 20.66,20.63, 20.59 (3 x - C ( O ) m ) .
endo-isomer:
1
H m : 65.39(dd, lH,J3,4=J4,5=9.8Hz, H-4),85.26(d,1H,JI,22.5Hz
,H-l), 6 5.20 (dd, lw J 2 , 3 4.0 &, H-3), 8 4.38 (d, IH, H-S), 6 4.26 (dd, IH, Js,6a4.8, J6a,
6i1
12.0 HZ,H-6a), 64.12(dd7 l H , J 5 , 6 b 2-6 HZ, H-6b), 6 3.70(ddd, lH, H-5), 6 3.49 (s,
3H, -C(CH3)0CH3), 6 2.12, 2.05,2.03 ( 3s, each 3 Y 3 x -C(O)Cfi), 6 1.52 (s, 3H,
-C(CH3)0CH3).
The triacetylated orthoester (Il)(2.01 g, 5.54 mmol) was dissolved m dry MeOH (46
ml). 1M NaOMeMeOH (320 pl) was added and the reaction mixture was stirred at room
temperature for 1 hr. The solvent was removed in vacuo to give a coIourless foam. This
crude triol was dissolved in DMF (16 ml) and cooled to O OC in an ice bath. NaH (1.12 g,
28.1 mmol, 5 equiv.) was added followed by BnBr (3.40 mi, 5 equiv.). The reaction
mixture was stirred at room temperature for 1.5 hr., aRer which the temperature was
lowered to O°C and the reaction quenched by adding dry MeOH (2.0 ml) dropwise. Once
H2 evolution subsided, the solvents and volatile materials were removed on a rotary
evaporator under high vacuum. The reaction mivture was partitioned behveen Et20 (60
ml) and H20 (20 ml). The aqueous layer was washed with Et20 (2 x 10 mi). The organic
fiactions were collected, washed with saturated NaCl (15 ml) and H20 (1 5 ml), dried over
anhydrous MgS04, fltered, and concentrated to yield the title compound (12) as a clear
wnip (2.61 g, 93%): [alo+27.6 O (C 0.98, CHCl3);
m.'"[alDc12.1
O
(c 1.65, CHC13)];
exo-isomer :
'H NiVR 6 7.41-7.23 (m,1 5 6 aromatic), 6 5.35 (d, lH, J I , 2.5 Hz ,H-l), 6
4.89, 4.60 (2d, 2H, J m 10.7 HZ, -CHaHbPh), 6 4.80,4.76 (2d72 Y AaHb
12.2 HZ,
-CHaHbPh), 6 4.60,4.54 (2d, 2H, J H12.1
~HZ> -CHaHbPh), 6 4.39 (dd, lH, J2,33.9 HZ,
H-Z), 6 3.92 (dd, IH,
J3,4 = J4,5 =
9.4 HZ, H-4), 6 3.76 (dd, lH, J5,6a 4.4, Jca,fjb 10.8 Hk,
H-6a), 6 3.72 (dd, lH, H-3), 6 3.70 (dd, lH, &,a 2.3 Hz, H-6b), 6 3.42 (ddd, lH, H-5),
S 3.28 (s, 3H, -C(CH3)0CH3), 6 1.73 (s, 3H, -C(CH3)OCH3).
I3cNMR:
6 138.24 (ZC), 137.85 (3 x aromatic C-1 of -CH2Ph), 6 128.50
(2C), 128.38 (2C), 128.29 (2C), 128.02 (4C), 127.98, 127.75, 127.51 (3C) (aromatic),
8 123.98 (-C(C&)OCH3), 6 97.55 (C- 1), 6 79.03 (C-3), 6 77.11 (C-2), 6 75.21, 73.36,
72.35 (3 x -CH2Ph), 6 74.19 (2C) ((2-4, C-5), 6 69.03 (C-6), 6 49.74 (-C(CH3)Oa),
S 24.37 (-C(CH3)0CH3).
endo-isomer :
1
H NMR: 6 7.41-7.23 (m, 15H, aromatic), 6 5.10 (d, lH, J I , 2.4 Hz,H-1),
64.91,4.63 ( 2 4 2H, J.& 10.7&-CHaHbPh),
- C H a P h ) , 6 4.5734.52 (2d, 2 Y J
H
64.83, 4.81 ( 2 4 2H, Jwm 12.1 Hz,
12.0
~
HZ,-CHaHbPh), 6 4. IO (dd, IH, J2,)4.1
HZ,H-2), 6 4.01 (dd, lH, J3,4= J4,s= 9.4 HZ, H-4), 6 3.76 (dd, lH, H-3), 6 3.74-3.67 (m,
H-Gb), 6 3.44-3.40 (qlH, H-5), 6 3.44 (s, 3H, -C(CH3)OCH3),6 1.56 (s, 3H,
2Y
-C(CH3)OC&).
The benzylated orthoester (12) (2.61 g, 5.15 mmol) was dissolved in 60 ml of dry MeOH.
Acetyl Chloride (1.80 ml) was added and the reaction mixture was s h e d at 76 OC under
an N2 atmosphere for 7.5 hr. The reaction was cooled to room temperature and the
MeOH removed in vacuo to yield a pale yellow glass. The residue was dissolved in
CH2Cl2 (50 ml), washed with saturated NaHC03 (3 x 50 ml) and H20 (50 ml), dried over
anhydrous NaS04, Gltered, and concentrated. The clear syrup was placed under high
vacuum to remove any residual solvent. Purification by flash column chromatography
(hex:EtOAc, 1: 1) gave the title compound (4) as a syrup (2.17 g, 9 1 %):
1.85, CH&);
[alr,+5 7.3
O
(c
@it.12' [a]*+59.7 O (c 1.85, CH2C12)];
1
HNMR: S4.80(d7 l H y J i , 2 1.7H~,H-1),64.82,4.50(2d,2Y
JHaHb
10.8H~,
-CHaHbPh), 6 4.70,4.66 (2d, 2-
JHaW
11.5 HZ, -CHaHbPh), 6 4.65, 4.54 (2d, 2% Jwaw
12.2 Hz,-CHaHbPh), 6 4.03 (dd, lH, J 2 , 3 2.8 HZ, H-2), 6 3.87 (dd, lH, J3,4 8.5 HZ,H-
3), 6 3.84 (dd, lH, 5 4 . 5 8.5 Hz,H-4), F 3.77-3.67 (m., 3H, H-5, B 6 a , H-6b), 6 3.36 (s,
3H, -OCH3).
13
C NMR: 6 138.36, 138.17, 137.94 (3 x aromatic C-1 of-CHzPh), 6 128.51
(ZC), 128.33 (4C), 127.87 (7C), 127.62, 127.59 (aromatic), 6 100.33 (C-l), 6 80.16,
74.27 (C-3, C-4), 6 75.05, 73.47,7 1-95(3 x -CH2Ph), 6 70.94, 68.98 (C-5, C-6),
6 68.29 (C-2), 6 54.87 (-0CH3).
The disaccharide 5 (379 mg, 0.5 1 m o l ) and the selectively protected methyl
mannopyranoside 4 (308 mg, 0.66 mrnol) were dissolved in dry CH2C12(15.00 ml). The
solution was stirred with freshly activated powdered 4 À molecular siwes (-0.60 g) under
an N2 atmosphere at room temperature for 20 minutes. N-iodosuccinimide (15 1 mg, 0.67
mmol, 1.3 equiv.) was added foilowed, after 10 minutes, by t r a c acid (5 pl) via syringe.
An immediate reaction to produce a dark purple-brown colour ensued. After 16 hr.,
triethyIamine was added to quench the reaction and the mixture was fltered and washed
with CH2C12 (2 x 20 ml). The filtrate was washed with 10 % aqueous NaS20s (2 x 45 ml)
and H20 (75 ml), dried over anhydrous MgS04, filtered and concentrated to a brown oil.
mirification by flash column chromatography (toluene-EtOAc, 3: 1) gave the desired
trisacchande (6) as a colourless foam (393 mg, 68 %): [a]- 36 O (c 0.25, CH2C12);
'H NMR (CD2Ch): 6 8.12-7.09 (m,25H, aromatic), 6 5.70 (dd, lH, JI*,1.5,
J2*,333.6 HZ, H-2'),
5"
3.4, J5.9,
&j"
7.5,
6 5.64 (s, 1- PhCH), 6 5.29 (d, 1H, H-lu), 6 5.25 (ddd, lH, J4-,
J5",6bW
H-5"), 6 5.24 (s, IH, H-l'), 6 4.91 (d, IH,
4.1
J2",3"
1.5
Hz, H-2"), 6 4.82 (dd, lH,J3",4" 5.6 Hz, H-3"), 6 4.80 (d, lH, J 1 , 2 1-9Hz,H-1), 6
4-8594.57 (2d, 2H, JEIaa
I l . 1 HZ, -CHaHbPh), 6 4.70, 4.66 (2d, 2K, JHa-
-CHaHbPh), 6 4.64,4.58 (2d, 2H,
11.8 HZ,
12.2 HZ,-CNaHbPh), 6 4.41 (dd, lH, J37,47
9.7 Hz, H-3'), 6 4.32 (dd, lH, Jy,6a' 4.1, J6ti9,6bp 10.2 HZ, H-6a'), 6 4.24 (dd, lH, J4-,53.3 Hz, H-4"), 6 4.11-4.01,III(
ZH, H-3, H-4'), 6 4.06 (dd, lH, J2,3
2.9 HZ, H-2), 6
4.05 (dd, lH, J&",
6bw 11.8 &, H-6a"),
6 3.96 (dd, lH, H-6b7'), 6 3.92-3.83 (m, 3H, H5 ' , H-6b7,H-4), 6 3.75 (m,3H, H-5, H-6a, H-6b), 6 3.37 (s, 3H, -OC&),
6 2.11, 2.08,
1-89, 1.82 (4s, each 3H, -C(O)Cfi).
"C NMR (CDzClZ): 6 170.19, 169.92, 169.80, 169.00 (4 x -C(0)CH3),
6 165.27 (-C(O)Ph), 6 138.47, 138.39, 138.16 (3 x aromatic C-1 of -C&Ph), 6137.41
(aromatic C- 1 of -C(O)Ph), 6 133.22 (aromatic C-1 of PhCH), 6 129.93 (SC), 129.84,
129.04, 128.59 (SC), 128.40 (2C), 128.32 (2C), 128.29 (SC), 128.23 (2C), 128.11 (4C),
127.92, 127.65, 127.55 (2C), 127.50, 126.00 (2C) (aromatic), 6 102.63 (C-l"),
6 101.80 (PhCH), 6 100.22 (C-l'), 6 99.78 (C-l), 6 80.80 (C-Z"), 6 80.37 (C-4'),
6 80.37, 74.92 (C-4, C-5), 6 77.17, 74.3 8, 64.28 (C-2, C-3, C-4'), 6 76.78 (C-3 "),
6 75.12, 73.34, 72.50 (3 x -CHZPh), S 71.83, 68.79 (C-5, C-6), 6 70.03 (C-3'), 6 69.53
(C-5'7, 6 69.27 (C-6), 6 69.02 (C-2'), 6 62.71 (C-6"), 6 54.72 (-0CH3),6 20.71
(2C), 20.54, 20.3 1 (4 x C ( 0 ) a ) .
Anal. Calc'd. for C62II68021: C, 64.80; H, 5.96. Found: C, 64.95; H, 5.91.
The protected trisaccharide (6) (129 mg, O. 11 m o l ) was dissolved in fieshly distilled
MeOH (25.00 ml). Gaseous NH3 was bubbled through the solution, while stirrhg under
an NI atmosphere for 18 hr. The reaction was concentrated by rotary-evaporation under a
hi& vacuum (- 0.05 Torr), then placed directly onto the high vacuum pump for 25 hr.
Purification by flash column chromatography (CHzC12:MeOH, 5: 1) gave the desired
partially deprotected trisaccharide as a colourless foam (87.1 mg, 89 %).
1
H NMR ( CD30D): 6 7.50-7.12 (m, 20Y aromatic), 6 5.60 (s, lH, PhCH), 6 5.13 (br s,
1 y H - l " ) , 6 5.09(br s, 1 y H - l ' ) , 64.18(d,
lHJ1.2
1.9)IZ.H-1),84.78,4.52(2d,
2H, J H , 1~1.0 Hz, -CHaHbPh), 6 4.69, 4.63 (2d, 2H, Jfim 11.6 HZ, -CHaHbPh), 6
4.65,4.56 (2d, 2H, Jbpb 12.3%
-CHaHbPh), 64.23-4.18, 64.07-3.96,63.89-3.78,
63.72-3.65 (4 m, 15H, H-2, H-2', H-2", H-3, H-3', H-3",H-4, H-4', H-4",.H-5, H-5',
4.8 HZ, H-5"),
H-6a, H-6b, H-6a', H-6b'), 6 3.63 (ddd, lH, J4.., 2.7, & 6 a 7 ' 7.5, J5p*,6b*v
599
6 3.47 (dd, lH, Jw,
st,-
1 1.2 Hz, H-6a"), 6 3.37 (dd, lH, H-6b"), 6 3.36 (s, 3H, 0CH3).
13
C NMR (CD30D): 6 139.75, 139.67, 139.58 (3 x aromatic C- 1 of -CH2Ph),
6 139.18 (aromatic C- 1 of -C(O)Ph), 6 129.84, 129.50 (2C), 129.47 (2C), 129.30 (2C),
129.09 (4C), 129.06 (4C), 128.83, 129.66 (ZC), 127.51 (2C) (aromatic), 6 106.66 (Cl''), 6 104.10 (C-l'), S 103.15 (PhCH), 6 101.38 (C-l), 6 86.06, 82.29, 80.91, 79.28,
78.41, 76.64,76.04, 73.22, 73.05, 70.13, 69.72,69.60, 65.89 (C-2, C-2', C-2", C-3, C3', C-3", C-4, C-4', C-4", C-5, C-5', C-6, C-6'), 8 75.97, 75.44,73.30 (3 x -Cl&Ph),
6 72.60 (C-5"), 6 64.82 (C-6"), 6 55.47 (-0CH3).
The partially deprotected trisaccharide (13) (75 mg, 0.09 mmol) was dissolved in 4: 1,
HOAc:H20 (10.00 ml) and stirred with Pd-C (1 14 mg) under H2 (52 psi). After 20 hr. the
reaction mixture was fltered through a pad of Celite and the Celite rinsed with distilled
Hz0 (4 x 20 ml). The combined mtrates were evaporated to clryness and the residue coevaporated several times with distîlied Hz0 to remove any traces of HOAc. The target
trisaccharide (1)was obtained as a colourless foam (4 mg, 96 %): [alo- 54.5 O (c 0.16,
H20);
1
HNMR(D20): 6 5.13 (d, l H , J i ~ ? , z 1.6
~ * Hz, H-l"), 6 5.03 (d, lH,
6 4.96 (d,
lK J 1 , 2
3-3.1 &H-2"),
1.5 HZ, H-17),
1.9 Hz, m l ) , 6 4.24 (dd, lH, J2*,3* 2.9 Hz, H-2'), 6 4.20 (dd, lH, J2-,
64.04(dd, l H , & , ~ ~ ~ 6HqH-3"),
.5
64.01 (dd, lH,
J47v,1.T
3.8
Hz,K4"), 6 3.93 (dd, IH, &., 3 3.3 % H-2), 6 3.89 (dd, lJ3, J3>, 9.4 HZ, H-3'), 6
*7
3.83 (dd, lH, J3, 9.7 Hz, H-3), 6 3.83-3.36 (m, 1lH, H-4, H-4', H-5,
H-5', H4", H-6a,
H-6b, H-6a7,H-6b', H-6a7', H-6b7'), 6 3.37 (s, 3H, -OCH3).
13
C NMR (D20): 6 107.02 (Col"), 6 104.73 (C-l'), 6 101.93(C-l), 6 85.50
(C-4"), 6 83.98 (C-2"), 6 81.27 (C-2), 6 79.63 (C-3"), 6 75.82, 75.70,75.35, 72.81,
69.60 (C-4, C-4', C-5, C-5', C-5")' 6 69.31 (C-2'), 6 67.80, 65.48,63.58 (C-6, C-6', C6"), 6 63.76 (C-3), 6 57.46 (-0CH3).
Anal. Calc'd. for C19&4016: C,44.02; H, 6.6 1. Fowd: C, 43.68; H, 6.30.
4. References
l
Horowitz, M.I. ;Pigman, W. The GIycoconjugates 1977-1982, Vol 1-IV, Academic
Press, New York.
Montreuil, J. A h . Carbohydr. Chem. Biochem. l98O,37, 157.
3
Singer, S. J. Annu. Rev. Biochem. 1974,43, 805.
4
Hakamori, S. Annu. Rev. Biochem. 1981,50, 733.
Li, Y.-T. ;Li, S. -C. A h . Carbohydr. Chem. Biochem. l98f,#O, 23 5.
Feia, T; Childs, R A. Biochenz. J 1987,245, 1 .
8
Lasky, L. A. Science 1992,258,964.
3
Macher, B. A. ; Stults, C. L. M. in Cell Surface and Extrocellular GZycoconjugates
Roberts, D. D.; Mecham, R P. eds., 1993, Academic Press, San Diego, p 223.
1O
Watkins, W.M. Science 1966, 152, 172.
Il
Roberis, D. D.;Mecham, R P. Cell Surface and Extracellular GIycoconjugutes
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